Compositions and methods for treating infections using analogues of indolicidin

ABSTRACT

Compositions and methods for treating infections, especially bacterial infections, are provided. Indolicidin peptide analogues containing at least two basic amino acids are prepared. The analogues are administered as modified peptides, preferably containing photo-oxidized solubilizer.

CROSS-RELATED APPLICATIONS

[0001] The present application claims priority from U.S. ProvisionalApplication No. 60/024,754, filed Aug. 21, 1996, and U.S. ProvisionalApplication No. 60/034,949, filed Jan. 13, 1997.

TECHNICAL FIELD

[0002] The present invention relates generally to treatment ofmicroorganism-caused infections, and more specifically, to compositionscomprising indolicidin analogues, polymer-modified analogues, and theiruses in treating infections.

BACKGROUND OF THE INVENTION

[0003] For most healthy individuals, infections are irritating, but notgenerally life-threatening. Many infections are successfully combated bythe immune system of the individual. Treatment is an adjunct and isgenerally readily available in developed countries. However, infectiousdiseases are a serious concern in developing countries and inimmunocompromised individuals.

[0004] In developing countries, the lack of adequate sanitation andconsequent poor hygiene provide an environment that fosters bacterial,parasitic, fungal and viral infections. Poor hygiene and nutritionaldeficiencies may diminish the effectiveness of natural barriers, such asskin and mucous membranes, to invasion by infectious agents or theability of the immune system to clear the agents. As well, a constantonslaught of pathogens may stress the immune system defenses of antibodyproduction and phagocytic cells (e.g., polymorphic neutrophils) tosubnormal levels. A breakdown of host defenses can also occur due toconditions such as circulatory disturbances, mechanical obstruction,fatigue, smoking, excessive drinking, genetic defects, AIDS, bone marrowtransplant, cancer, and diabetes. An increasingly prevalent problem inthe world is opportunistic infections in individuals who are HIVpositive.

[0005] Although vaccines may be available to protect against some ofthese organisms, vaccinations are not always feasible, due to factorssuch as inadequate delivery mechanisms and economic poverty, oreffective, due to factors such as delivery too late in the infection,inability of the patient to mount an immune response to the vaccine, orevolution of the pathogen. For other pathogenic agents, no vaccines areavailable. When protection against infection is not possible, treatmentof infection is generally pursued. The major weapon in the arsenal oftreatments is antibiotics. While antibiotics have proved effectiveagainst many bacteria and thus saved countless lives, they are not apanacea. The overuse of antibiotics in certain situations has promotedthe spread of resistant bacterial strains. And of great importance,antibacterials are useless against viral infections.

[0006] A variety of organisms make cationic (positively charged)peptides, molecules used as part of a non-specific defense mechanismagainst microorganisms. When isolated, these peptides are toxic to awide variety of microorganisms, including bacteria, fungi, and certainenveloped viruses. One cationic peptide found in neutrophils isindolicidin. While indolicidin acts against many pathogens, notableexceptions and varying degrees of toxicity exist.

[0007] Although cationic peptides show efficacy in vitro against avariety of pathogenic cells including gram-positive bacteria,gram-negative bacteria, and fungi, these peptides are generally toxic tomammals when injected, and therapeutic indices are usually quite small.Approaches to reducing toxicity have included development of aderivative or delivery system that masks structural elements involved inthe toxic response or that improves the efficacy at lower doses. Otherapproaches under evaluation include liposomes and micellular systems toimprove the clinical effects of peptides, proteins, and hydrophobicdrugs, and cyclodextrins to sequester hydrophobic surfaces duringadministration in aqueous media. For example, attachment of polyethyleneglycol (PEG) polymers, most often by modification of amino groups,improves the medicinal value of some proteins such as asparaginase andadenosine deaminase, and increases circulatory half-lives of peptidessuch as interleukins.

[0008] None of these approaches are shown to improve administration ofcationic peptides. For example, methods for the stepwise synthesis ofpolysorbate derivatives that can modify peptides by acylation reactionshave been developed, but acylation alters the charge of a modifiedcationic peptide and frequently reduces or eliminates the antimicrobialactivity of the compound. Thus, for delivery of cationic peptides, aswell as other peptides and proteins, there is a need for a systemcombining the properties of increased circulatory half-lives with theability to form a micellular structure.

[0009] The present invention discloses analogues of indolicidin,designed to broaden its range and effectiveness, and further provideother related advantages. The present invention also provides methodsand compositions for modifying peptides, proteins, antibiotics and thelike to reduce toxicity, as well as providing other advantages.

SUMMARY OF THE INVENTION

[0010] The present invention generally provides indolicidin analogues.In related aspects, an indolicidin analogue is provided, comprising upto 25 amino acids and containing the formula: RXZXXZXB; BXZXXZXB whereinat least one Z is valine; BBBXZXXZXB; BXZXXZXBBB_(n)(AA)_(n)MILBBAGS;BXZXXZXBB(AA)_(n)M; LBB_(n)XZ_(n)XXZ_(n)XRK; LK_(n)XZXXZXRRK;BBXZXXZXBBB, wherein at least two X residues are phenylalanine;BBXZXXZXBBB, wherein at least two X residues are tyrosine; and wherein Zis proline or valine; X is a hydrophobic residue; B is a basic aminoacid; AA is any amino acid, and n is 0 or 1. In preferred embodiments, Zis proline, X is tryptophan and B is arginine or lysine. In otheraspects, indolicidin analogues having specific sequences are provided.In certain embodiments, the indolicidin analogues are coupled to form abranched peptide. In other embodiments, the analogue has one or moreamino acids altered to a corresponding D-amino acid, and in certainpreferred embodiments, the N-terminal and/or the C-terminal amino acidis a D-amino acid. Other preferred modifications include analogues thatare acetylated at the N-terminal amino acid, amidated at the C-terminalamino acid, esterified at the C-terminal amino acid, modified byincorporation of homoserine/homoserine lactone at the C-terminal aminoacid, and conjugated with polyethylene glycol or derivatives thereof.

[0011] In other aspects, the invention provides an isolated nucleic acidmolecule whose sequence comprises one or more coding sequences of theindolicidin analogues, expression vectors, and host cells transfected ortransformed with the expression vector.

[0012] Other aspects provide a pharmaceutical composition comprising atleast one indolicidin analogue and a physiologically acceptable buffer,optionally comprising an antibiotic agent. Preferred combinationsinclude I L K K F P F F P F R R K and Ciprofloxacin; I L K K F P F F P FR R K and Mupirocin; I L K K Y P Y Y P Y R R K and Mupirocin; I L K K WP W W P W R K and Mupirocin; I L R R W P W W P W R R R and Piperacillin;W R I W K P K W R L P K W and Ciprofloxacin; W R I W K P K W R L P K Wand Mupirocin; W R I W K P K W R L P K W and Piperacillin; I L R W V W WV W R R K and Piperacillin; and I L K K W P W W P W K and Mupirocin. Inother embodiments, the pharmaceutical composition further comprises anantiviral agent, (e.g., acyclovir; amantadine hydrochloride; didanosine;edoxudine; famciclovir; foscarnet; ganciclovir; idoxuridine; interferon;lamivudine; nevirapine; penciclovir; podophyllotoxin; ribavirin;rimantadine; sorivudine; stavudine; trifluridine; vidarabine;zalcitabine and zidovudine); an antiparasitic agent (e.g.,8-hydroxyquinoline derivatives; cinchona alkaloids; nitroimidazolederivatives; piperazine derivatives; pyrimidine derivatives andquinoline derivatives, albendazole; atovaquone; chloroquine phosphate;diethylcarbamazine citrate; eflornithine; halofantrine; iodoquinol;ivermectin; mebendazole; mefloquine hydrochloride; melarsoprol B;metronidazole; niclosamide; nifurtimox; paromomycin; pentamidineisethionate; piperazine; praziquantel; primaquine phosphate; proguanil;pyrantel pamoate; pyrimethamine; pyrvinium pamoate; quinidine gluconate;quinine sulfate; sodium stibogluconate; suramin and thiabendazole); anantifungal agent (e.g., allylamines; imidazoles; pyrimidines andtriazoles, 5-fluorocytosine; amphotericin B; butoconazole;chlorphenesin; ciclopirox; clioquinol; clotrimazole; econazole;fluconazole; flucytosine; griseofulvin; itraconazole; ketoconazole;miconazole; naftifine hydrochloride; nystatin; selenium sulfide;sulconazole; terbinafine hydrochloride; terconazole; tioconazole;tolnaftate and undecylenate). In yet other embodiments, the compositionis incorporated in a liposome or a slow-release vehicle.

[0013] In yet another aspect, the invention provides a method oftreating an infection, comprising administering to a patient atherapeutically effective amount of a pharmaceutical composition. Theinfection may be caused by, for example, a microorganism, such as abacterium (e.g.,Gram-negative or Gram-positive bacterium or anaerobe;examples are Acinetobacter spp., Enterobacter spp., E. coli, H.influenzae, K. pneumoniae, P. aeruginosa, S. marcescens and S.maltophilia, Bordetella pertussis; Brucella spp.; Campylobacter spp.;Haemophilus ducreyi; Helicobacter pylori; Legionella spp.; Moraxellacatarrhalis; Neisseria spp.; Salmonella spp.; Shigella spp. and Yersiniaspp.; E. faecalis, S. aureus, E. faecium, S. pyogenes, S. pneumoniae andcoagulase-negative staphylococci; Bacillus spp.; Corynebacterium spp.;Diphtheroids; Listeria spp. and Viridans Streptococci.; Clostridiumspp., Bacteroides spp. and Peptostreptococcus spp.; Borrelia spp.;Chlamydia spp.; Mycobacterium spp.; Mycoplasma spp.; Propionibacteriumacne; Rickettsia spp.; Treponema spp. and Ureaplasma spp.) fungus(e.g.,yeast and/or mold), parasite (e.g., protozoan, nematode, cestodeand trematode, such as Babesia spp.; Balantidium coli; Blastocystishominis; Cryptosporidium parvum; Encephalitozoon spp.; Entamoeba spp.;Giardia lamblia; Leishmania spp.; Plasmodium spp.; Toxoplasma gondii;Trichomonas spp. Trypanosoma spp, Ascaris lumbricoides; Clonorchissinensis; Eehinococcus spp.; Fasciola hepatic, Fasciolopsis buski;Heterophyes heterophyes; Hymenolepis spp.; Schistosoma spp.; Taenia spp.and Trichinella spiralis) or virus (e.g., Alphavirus; Arenavirus;Bunyavirus; Coronavirus; Enterovirus; Filovirus; Flavivirus; Hantavirus;HTLV-BLV; Influenzavirus; Lentivirus; Lyssavirus; Paramyxovirus;Reovirus; Rhinovirus and Rotavirus, Adenovirus; Cytomegalovirus;Hepadnavirus; Molluscipoxvirus; Orthopoxvirus; Papillomavirus;Parvovirus; Polyomavirus; Simplexvirus and Varicellovirus).

[0014] In other aspects, a composition is provided, comprising anindolicidin analogue and an antibiotic. In addition, a device, which maybe a medical device, is provided that is coated with the indolicidinanalogue and may further comprise an antibiotic agent.

[0015] In other aspects, antibodies that react specifically with any oneof the analogues described herein are provided. The antibody ispreferably a monoclonal antibody or single chain antibody.

[0016] In a preferred aspect, the invention provides a compositioncomprising a compound modified by derivatization of an amino group witha conjugate comprising activated polyoxyalkylene glycol and a fattyacid. In preferred embodiments, the conjugate further comprises sorbitanlinking the polyoxyalkylene glycol and fatty acid, and more preferablyis polysorbate. In preferred embodiments, the fatty acid is from 12-18carbons, and the polyoxyalkylene glycol is polyoxyethylene, such as witha chain length of from 2 to 100. In certain embodiments, the compound isa peptide or protein, such as a cationic peptide (e.g., indolicidin oran indolicidin analogue). In preferred embodiments, the polyoxyalkyleneglycol is activated by irradiation with ultraviolet light.

[0017] The invention also provides a method of making a compoundmodified with a conjugate of an activated polyoxyalkylene glycol and afatty acid, comprising: (a) freezing a mixture of the conjugate of anactivated polyoxyalkylene glycol and fatty acid with the compound; and(b) lyophilizing the frozen mixture; wherein the compound has a freeamino group. In preferred embodiments, the compound is a peptide orantibiotic. In other preferred embodiments, the mixture in step (a) isin an acetate buffer. In a related aspect, the method comprises mixingthe conjugate of an activated polyoxyalkylene glycol and fatty acid withthe compound; for a time sufficient to form modified compounds, whereinthe mixture is in a carbonate buffer having a pH greater than 8.5 andthe compound has a free amino group. The modified compound may beisolated by reversed-phase HPLC and/or precipitation from an organicsolvent.

[0018] The invention also provides a pharmaceutical compositioncomprising at least one modified compound and a physiologicallyacceptable buffer. and in certain embodiments, further comprises anantibiotic agent, antiviral agent, an antiparasitic agent, and/orantifungal agent. The composition may be used to treat an infection,such as those caused by a microorganism (e.g., bacterium, fungus,parasite and virus).

[0019] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, various references are set forth belowwhich describe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is an SDS-PAGE showing the extraction profile of inclusionbodies (ib) from whole cells containing MBI-11 fusion protein. Thefusion protein band is indicated by the arrow head. Lane 1, proteinstandards; lane 2, total lysate of XL1 Blue without plasmid; lane 3,total lysate of XL1 Blue (pR2h-11, pGP1-2), cultivated at 30° C.; lane4, total lysate of XL1 Blue (pR2h-11, pGP1-2), induced at 42° C.; lane5, insoluble fraction of inclusion bodies after Triton X100 wash; lane6, organic extract of MBI-11 fusion protein; lane 7, concentratedmaterial not soluble in organic extraction solvent.

[0021]FIG. 2 is an SDS-PAGE showing the expression profile of the MBI-11fusion protein using plasmid pPDR2h-11. Lane 1, protein standards; lane2, organic solvent extracted MBI-11; lane 3, total lysate of XL1 Blue(pPDR2h-11, pGP1-2), cultured at 30° C.; lane 4, total lysate of XL1Blue (pPDR2h-11, pGP1-2), induced at 42° C.

[0022]FIG. 3 presents time kill assay results for MBI 11CN, MBI 11F4CNand MBI 11B7CN. The number of colony forming units×10⁻⁴ is plottedversus time.

[0023]FIG. 4 is a graph presenting the extent of solubility of MBI 11CNpeptide in various buffers.

[0024]FIG. 5 is a reversed phase HPLC profile of MBI 11CN in formulationC1 (left graph panel) and formulation D (right graph panel).

[0025]FIG. 6 presents CD spectra of MBI 11CN and MBI 11B7CN.

[0026]FIG. 7 presents results of ANTS/DPX dye release of egg PCliposomes at various ratios of lipid to protein.

[0027]FIG. 8 presents graphs showing the activity of MBI 11B7CN againstmid-log cells grown in terrific broth (TB) or Luria-Bretani broth (LB).

[0028]FIG. 9 shows results of treatment of bacteria with MBI 10CN, MBI11CN, or a control peptide alone or in combination with valinomycin.

[0029]FIG. 10 is a graph showing treatment of bacteria with MBI 11B7CNin the presence of NaCl or Mg²⁺.

[0030]FIG. 11 is a graph presenting the in vitro amount of free MBI 11CNin plasma over time. Data is shown for peptide in formulation C1 andformulation D.

[0031]FIG. 12 is a graph presenting change in in vivo MBI 11CN levels inblood at various times after intravenous injection.

[0032]FIG. 13 is a graph presenting change in in vivo MBI 11CN levels inplasma at various times after intraperitoneal injection.

[0033]FIG. 14 is a graph showing the number of animals surviving an MSSAinfection after intraperitoneal injection of MBI 10CN, ampicillin, orvehicle.

[0034]FIG. 15 is a graph showing the number of animals surviving an MSSAinfection after intraperitoneal injection of MBI 11CN, ampicillin, orvehicle.

[0035]FIG. 16 is a graph showing the results of in vivo testing ofMBI-11A1CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0036]FIG. 17 is a graph showing the results of in vivo testing ofMBI-11E3CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0037]FIG. 18 is a graph showing the results of in vivo testing of:MBI-11F3CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0038]FIG. 19 is a graph showing the results of in vivo testing ofMBI-11G2CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0039]FIG. 20 is a graph showing the results of in viva testing ofMBI-11CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0040]FIG. 21 is a graph showing the results of in vivo testing ofMBI-11B1CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0041]FIG. 22 is a graph showing the results of in vivo testing ofMBI-11B7CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0042]FIG. 23 is a graph showing the results of in vivo testing ofMBI-11B8CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0043]FIG. 24 is a graph showing the results of in vivo testing ofMBI-11G4CN against S. aureus (Smith). Formulated peptide at variousconcentrations is administered by ip injection one hour after infectionwith S. aureus (Smith) by ip injection.

[0044]FIGS. 25A and B display a graph showing the number of animalssurviving an S. epidermidis infection after intravenous injection of MBI10CN, gentamicin, or vehicle. Panel A, i.v. injection 15 minpost-infection; panel B, i.v. injection 60 min post-infection.

[0045]FIG. 26 is a graph showing the number of animals surviving an MRSAinfection mice after intravenous injection of MBI 11CN, gentamicin, orvehicle.

[0046]FIG. 27 presents RP-HPLC traces analyzing samples for APS-peptideformation after treatment of activated polysorbate with a reducingagent. APS-MBI-11CN peptides are formed via lyophilization in 200 mMacetic acid-NaOH, pH 4.6, 1 mg/ml MBI 11CN, and 0.5% activatedpolysorbate 80. The stock solution of activated 2.0% polysorbate istreated with (a) no reducing agent, (b) 150 mM 2-mercaptoethanol, or (c)150 mM sodium borohydride for 1 hour immediately before use.

[0047]FIG. 28 presents RP-HPLC traces monitoring the formation ofAPS-MBI 11CN over time in aqueous solution. The reaction occurs in 200mM sodium carbonate buffer pH 10.0, 1 mg/ml MBI 11CN, 0.5% activatedpolysorbate 80. Aliquots are removed from the reaction vessel at theindicated time points and immediately analyzed by RP-HPLC.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms that areused herein.

[0049] The amino acid designations herein are set forth as either thestandard one- or three-letter code. A capital letter indicates an L-formamino acid; a small letter indicates a D-form amino acid.

[0050] As used herein, “indolicidin” refers to an antimicrobial cationicpeptide. Indolicidins may be isolated from a variety of organisms. Oneindolicidin is isolated from bovine neutrophils and is a 13 amino acidpeptide amidated at the carboxy-terminus in its native form (Selsted etal., J. Biol. Chem. 267:4292, 1992). An amino acid sequence ofindolicidin is presented in SEQ ID NO: 1.

[0051] As used herein, a “peptide analogue”, “analogue”, or “variant” ofindolicidin is at least 5 amino acids in length, has at least one basicamino acid (e.g., arginine and lysine) and has anti-microbial activity.Unless otherwise indicated, a named amino acid refers to the L-form,Basic amino acids include arginine, lysine, and derivatives. Hydrophobicresidues include tryptophan, phenylalanine, isoleucine, leucine, valine,and derivatives.

[0052] Also included within the scope of the present invention are aminoacid derivatives that have been altered by chemical means, such asmethylation (e.g., α methylvaline), amidation, especially of theC-terminal amino acid by an alkylamine (e.g., ethylamine, ethanolamine,and ethylene diamine) and alteration of an amino acid side chain, suchas acylation of the ε-amino group of lysine. Other amino acids that maybe incorporated in the analogue include any of the D-amino acidscorresponding to the 20 L-amino acids commonly found in proteins, iminoamino acids, rare amino acids, such as hydroxylysine, or non-proteinamino acids, such as homoserine and ornithine. A peptide analogue mayhave none or one or more of these derivatives, and D-amino acids. Inaddition, a peptide may also be synthesized as a retro-, inverto- orretro-inverto-peptide.

[0053] A. Indolicidin Analogues

[0054] As noted above, the present invention provides indolicidinanalogues. These analogues may be synthesized by chemical methods,especially using an automated peptide synthesizer, or produced byrecombinant methods. The choice of an amino acid sequence is guided by ageneral formula presented herein.

[0055] 1. Peptide Characteristics

[0056] The present invention provides indolicidin analogues. Theanalogues are at least 5 or 7 amino acids in length and preferably notmore than 15, 20, 25, 27, 30, or 35 amino acids. Analogues from 9 to 14residues are preferred.

[0057] General formulas for peptide analogues in the scope of thepresent invention may be set forth as:        RXZXXZXB (1)       BXZXXZXB (2)       BBBXZXXZXB (3) BXZXXZXBBB_(n)(AA)_(n)MILBBAGS(4)     BXZXXZXBB(AA)_(n)M (5)      LBB_(n)XZ_(n)XXZ_(n)XRK (6)      LK_(n)XZXXZXRRK (7)       BBXZXXZXBBB (8)       BBXZXXZXBBB (9)

[0058] wherein standard single letter amino abbreviations are used and;Z is proline, glycine or a hydrophobic residue, and preferably Z isproline or valine; X is a hydrophobic residue, such as tryptophan,phenylalanine, isoleucine, leucine and valine, and preferablytryptophan; B is a basic amino acid, preferably arginine or lysine; AAis any amino acid, and n is 0 or 1. In formula (2), at least one Z isvaline; in formula (8), at least two Xs are phenylalanine; and informula (9), at least two Xs are tyrosine. Additional residues may bepresent at the N-terminus, C-terminus, or both.

[0059] As described above, modification of any of the residues includingthe N- or C-terminus is within the scope of the invention. A preferredmodification of the C-terminus is amidation. Other modifications of theC-terminus include esterification and lactone formation. N-terminalmodifications include acetylation, acylation, alkylation, PEGylation,myristylation, and the like. Additionally, the peptide may be modifiedto form an APS-peptide as described below. The peptides may also belabeled, such as with a radioactive label, a fluorescent label, a massspectrometry tag, biotin and the like.

[0060] 2. Peptide Synthesis

[0061] Peptide analogues may be synthesized by standard chemicalmethods, including synthesis by automated procedure. In general, peptideanalogues are synthesized based on the standard solid-phase Fmocprotection strategy with HATU as the coupling agent. The peptide iscleaved from the solid-phase resin with trifluoroacetic acid containingappropriate scavengers, which also deprotects side chain functionalgroups. Crude peptide is further purified using preparativereversed-phase chromatography. Other purification methods, such aspartition chromatography, gel filtration, gel electrophoresis, orion-exchange chromatography may be used.

[0062] Other synthesis techniques, known in the art, such as the tBocprotection strategy, or use of different coupling reagents or the likecan be employed to produce equivalent peptides.

[0063] Peptides may be synthesized as a linear molecule or as branchedmolecules. Branched peptides typically contain a core peptide thatprovides a number of attachment points for additional peptides. Lysineis most commonly used for the core peptide because it has one carboxylfunctional group and two (alpha and epsilon) amine functional groups.Other diamino acids can also be used. Preferably, either two or threelevels of geometrically branched lysines are used; these cores form atetrameric and octameric core structure, respectively (Tam, Proc. Natl.Acad. Sci. USA 85:5409, 1988). Schematically, examples of these coresare represented as shown:

[0064] The attachment points for the peptides are typically at theircarboxyl functional group to either the alpha or epsilon amine groups ofthe lysines. To synthesize these multimeric peptides, the solid phaseresin is derivatized with the core matrix, and subsequent synthesis andcleavage from the resin follows standard procedures. The multimericpeptide is typically then purified by dialysis against 4 M guanidinehydrochloride then water, using a membrane with a pore size to retainonly multimers. The multimeric peptides may be used within the contextof this invention as for any of the linear peptides and are preferredfor use in generating antibodies to the peptides.

[0065] 3. Recombinant Production of Peptides

[0066] Peptide analogues may alternatively be synthesized by recombinantproduction (see e.g., U.S. Pat. No. 5,593,866). A variety of hostsystems are suitable for production of the peptide analogues, includingbacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae), insect(e.g., Sf9), and mammalian cells (e.g, CHO, COS-7). Many expressionvectors have been developed and are available for each of these hosts.Generally, bacteria cells and vectors that are functional in bacteriaare used in this invention. However, at times, it may be preferable tohave vectors that are functional in other hosts. Vectors and proceduresfor cloning and expression in E. coli are discussed herein and, forexample, in Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1987) andin Ausubel et al. (Current Protocols in Molecular Biology, GreenePublishing Co., 1995).

[0067] A DNA sequence encoding one or more indolicidin analogues isintroduced into an expression vector appropriate for the host. Inpreferred embodiments, the analogue gene is cloned into a vector tocreate a fusion protein. The fusion partner is chosen to contain ananionic region, such that a bacterial host is protected from the toxiceffect of the peptide. This protective region effectively neutralizesthe antimicrobial effects of the peptide and also may prevent peptidedegradation by host proteases. The fusion partner (carrier protein) ofthe invention may further function to transport the fusion peptide toinclusion bodies, the periplasm, the outer membrane, or theextracellular environment. Carrier proteins suitable in the context ofthis invention specifically include, but are not limited to,glutathione-S-transferase (GST), protein A from Staphylococcus aureus,two synthetic IgG-binding domains (ZZ) of protein A, outer membraneprotein F, β-galactosidase (lacZ), and various products of bacteriophageλ and bacteriophage T7. From the teachings provided herein, it isapparent that other proteins may be used as carriers. Furthermore, theentire carrier protein need not be used, as long as the protectiveanionic region is present. To facilitate isolation of the peptidesequence, amino acids susceptible to chemical cleavage (e.g., CNBr) orenzymatic cleavage (e.g., V8 protease, trypsin) are used to bridge thepeptide and fusion partner. For expression in E. coli, the fusionpartner is preferably a normal intracellular protein that directsexpression toward inclusion body formation. In such a case, followingcleavage to release the final product, there is no requirement forrenaturation of the peptide. In the present invention, the DNA cassette,comprising fusion partner and peptide gene, may be inserted into anexpression vector, which can be a plasmid, virus or other vehicle knownin the art. Preferably, the expression vector is a plasmid that containsan inducible or constitutive promoter to facilitate the efficienttranscription of the inserted DNA sequence in the host. Transformationof the host cell with the recombinant DNA may be carried out byCa⁺⁺s-mediated techniques, by electroporation, or other methods wellknown to those skilled in the art.

[0068] Briefly, a DNA fragment encoding a peptide analogue is derivedfrom an existing cDNA or genomic clone or synthesized. A convenientmethod is amplification of the gene from a single-stranded template. Thetemplate is generally the product of an automated oligonucleotidesynthesis. Amplification primers are derived from the 5′ and 3′ ends ofthe template and typically incorporate restriction sites chosen withregard to the cloning site of the vector. If necessary, translationalinitiation and termination codons can be engineered into the primersequences. The sequence encoding the protein may be codon-optimized forexpression in the particular host. Thus, for example, if the analoguefusion protein is expressed in bacteria, codons are optimized forbacterial usage. Codon optimization is accomplished by automatedsynthesis of the entire gene or gene region, ligation of multipleoligonucleotides, mutagenesis of the native sequence, or othertechniques known to those in the art.

[0069] At minimum, the expression vector should contain a promotersequence. However, other regulatory sequences may also be included. Suchsequences include an enhancer, ribosome binding site, transcriptiontermination signal sequence, secretion signal sequence, origin ofreplication, selectable marker, and the like. The regulatory sequencesare operationally associated with one another to allow transcription andsubsequent translation. In preferred aspects, the plasmids used hereinfor expression include a promoter designed for expression of theproteins in bacteria. Suitable promoters, including both constitutiveand inducible promoters, are widely available and are well known in theart. Commonly used promoters for expression in bacteria includepromoters from T7, T3, T5, and SP6 phages, and the trp, lpp, and lacoperons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tacand trc, may also be used.

[0070] In preferred embodiments, the vector includes a transcriptionterminator sequence. A “transcription terminator region” is a sequencethat provides a signal that terminates transcription by the polymerasethat recognizes the selected promoter. The transcription terminator maybe obtained from the fusion partner gene or from another gene, as longas it is functional in the host.

[0071] Within a preferred embodiment, the vector is capable ofreplication in bacterial cells. Thus, the vector may contain a bacterialorigin of replication. Preferred bacterial origins of replicationinclude f1-ori and col E1 ori, especially the ori derived from pUCplasmids. Low copy number vectors (e.g., pPD100) may also be used,especially when the product is deleterious to the host.

[0072] The plasmids also preferably include at least one selectablemarker that is functional in the host. A selectable marker gene confersa phenotype on the host that allows transformed cells to be identifiedand/or selectively grown. Suitable selectable marker genes for bacterialhosts include the chloroamphenicol resistance gene (Cm^(r)), ampicillinresistance gene (Amp^(r)), tetracycline resistance gene (Tc^(r))kanamycin resistance gene (Kan^(r)), and others known in the art. Tofunction in selection, some markers may require a complementarydeficiency in the host.

[0073] In some aspects, the sequence of nucleotides encoding the peptideanalogue also encodes a secretion signal, such that the resultingpeptide is synthesized as a precursor protein, which is subsequentlyprocessed and secreted. The resulting secreted protein may be recoveredfrom the periplasmic space or the fermentation medium. Sequences ofsecretion signals suitable for use are widely available and are wellknown (von Heijne, J. Mol. Biol. 184:99-105, 1985).

[0074] The vector may also contain a gene coding for a repressorprotein, which is capable of repressing the transcription of a promoterthat contains a repressor binding site. Altering the physiologicalconditions of the cell can depress the promoter. For example, a moleculemay be added that competitively binds the repressor, or the temperatureof the growth media may be altered. Repressor proteins include, but arenot limited to the E. coli lacI repressor (responsive to induction byIPTG), the temperature sensitive λcI857 repressor, and the like.

[0075] Examples of plasmids for expression in bacteria include the pETexpression vectors pET3a, pET 11a, pET 12a-c, and pET 15b (see U.S. Pat.No. 4,952,496; available from Novagen, Madison, Wis.). Low copy numbervectors (e.g., pPD100) can be used for efficient overproduction ofpeptides deleterious to the E. coli host (Dersch et al., FEMS Microbiol.Lett. 123: 19, 1994).

[0076] Bacterial hosts for the T7 expression vectors may containchromosomal copies of DNA encoding T7 RNA polymerase operably linked toan inducible promoter (e.g., lacUV promoter; see, U.S. Pat. No.4,952,496), such as found in the E. coli strains HMS174(DE3)pLysS,BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3). T7 RNA polymerase can also bepresent on plasmids compatible with the T7 expression vector. Thepolymerase may be under control of a lambda promoter and repressor(e.g., pGP1-2; Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82:1074, 1985).

[0077] The peptide analogue protein is isolated by standard techniques,such as affinity, size exclusion, or ionic exchange chromatography, HPLCand the like. An isolated peptide should preferably show a major band byCoomassie blue stain of SDS-PAGE that is at least 90% of the material.

[0078] 4. Generation of Analogues by Amplification-Based Semi-RandomMutagenesis

[0079] Indolicidin analogues can be generated using an amplification(e.g., PCR)-based procedure in which primers are designed to targetsequences at the 5′ and 3′ ends of an encoded parent peptide, forexample indolicidin. Amplification conditions are chosen to facilitatemisincorporation of nucleotides by the thermostable polymerase duringsynthesis. Thus, random mutations are introduced in the originalsequence, some of which result in amino acid alteration(s).Amplification products may be cloned into a coat protein of a phagevector, such as a phagemid vector, packaged and amplified in anacceptable host to produce a display library.

[0080] These libraries can then be assayed for antibiotic activity ofthe peptides. Briefly, bacteria infected with the library are plated,grown, and overlaid with agarose containing a bacterial strain that thephage are unable to infect. Zones of growth inhibition in the agaroseoverlay are observed in the area of phage expressing an analogue withanti-bacterial activity. These inhibiting phage are isolated and thecloned peptide sequence determined by DNA sequence analysis. The peptidecan then be independently synthesized and its antibiotic activityfurther investigated.

[0081] 5. Antibodies to Indolicidin Analogues

[0082] Antibodies are typically generated to a specific peptide analogueusing multiple antigenic peptides (MAPs) that contain approximatelyeight copies of the peptide linked to a small non-immunogenic peptidylcore to form an immunogen. (See, in general, Harlow and Lane, supra.)The MAPs are injected subcutaneously into rabbits or into mice or otherrodents, where they may have sufficiently long half-lives to facilitateantibody production. After twelve weeks blood samples are taken, serumis separated and tested in an ELISA assay against the original peptide,with a positive result indicating the presence of antibodies specific tothe target peptide. This serum can then be stored and used in ELISAassays to specifically measure the amount of the specific analogue.Alternatively, other standard methods of antibody production may beemployed, for example generation of monoclonal antibodies.

[0083] Within the context of the present invention, antibodies areunderstood to include monoclonal antibodies, polyclonal antibodies,anti-idiotypic antibodies, antibody fragments (e.g., Fab, and F(ab′)₂,F_(v) variable regions, or complementarity determining regions).Antibodies are generally accepted as specific against indolicidinanalogues if they bind with a K_(d) of greater than or equal to 10⁻⁷M,preferably greater than of equal to 10⁻⁸M. The affinity of a monoclonalantibody or binding partner can be readily determined by one of ordinaryskill in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949).Once suitable antibodies have been obtained, they may be isolated orpurified by many techniques well known to those of ordinary skill in theart.

[0084] Monoclonal antibodies may also be readily generated fromhybridoma cell lines using conventional techniques (see U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988). Briefly, within one embodiment, a subject animal such as arat or mouse is injected with peptide, generally administered as anemulsion in an adjuvant such as Freund's complete or incomplete adjuvantin order to increase the immune response. The animal is generallyboosted at least once prior to harvest of spleen and/or lymph nodes andimmortalization of those cells. Various immortalization techniques, suchas mediated by Epstein-Barr virus or fusion to produce a hybridoma, maybe used. In a preferred embodiment, immortilization occurs by fusionwith a suitable myeloma cell line to create a hybridoma that secretesmonoclonal antibody. Suitable myeloma lines include, for example, NS-1(ATCC No. TIB 18), and P3X63-Ag 8.653 (ATCC No. CRL 1580). The preferredfusion partners do not express endogenous antibody genes. After aboutseven days, the hybridomas may be screened for the presence ofantibodies that are reactive against a telomerase protein. A widevariety of assays may be utilized (see Antibodies: A Laboratory Manual,Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).

[0085] Other techniques may also be utilized to construct monoclonalantibodies (see Huse et al., Science 246:1275-1281, 1989; Sastry et al.,Proc. Natl. Acad. Sci. USA 86:5728-5732, 1989; Alting-Mees et al.,Strategies in Molecular Biology 3:1-9, 1990; describing recombinanttechniques). These techniques include cloning heavy and light chainimmunoglobulin cDNA in suitable vectors, such as λImmunoZap(H) andλImmunoZap(L). These recombinants may be screened individually orco-expressed to form Fab fragments or antibodies (see Huse et al.,supra; Sastry et al., supra). Positive plaques may subsequently beconverted to a non-lytic plasmid that allows high level expression ofmonoclonal antibody fragments from E. coli.

[0086] Similarly, portions or fragments, such as Fab and Fv fragments,of antibodies may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to yield isolated variableregions of an antibody. Within one embodiment, the genes which encodethe variable region from a hybridoma producing a monoclonal antibody ofinterest are amplified using nucleotide primers for the variable region.In addition, techniques may be utilized to change a “murine” antibody toa “human” antibody, without altering the binding specificity of theantibody.

[0087] B. Testing

[0088] Indolicidin analogues of the present invention are assessedeither alone or in combination with an antibiotic agent or anotheranalogue for their potential as antibiotic therapeutic agents using aseries of assays. Preferably, all peptides are initially assessed invitro, the most promising candidates selected for further assessment invivo, and using the results of these assays candidates are selected forpre-clinical studies. The in vitro assays include measurement ofantibiotic activity, toxicity, solubility, pharmacology, secondarystructure, liposome permeabilization and the like. In vivo assaysinclude assessment of efficacy in animal models, antigenicity, toxicity,and the like. In general, in vitro assays are initially performed,followed by in vivo assays.

[0089] 1. In vitro Assays

[0090] Indolicidin analogues are assessed for antibiotic activity by anassay such as an agarose dilution MIC assay or a broth dilution ortime-kill assay. Antibiotic activity is measured as inhibition of growthor killing of a microorganism (e.g., bacteria, fungi). Briefly, acandidate analogue in Mueller Hinton broth supplemented with calcium andmagnesium is mixed with molten agarose. Other formulations of broths andagars may be used as long as the peptide analogue can freely diffusethrough the medium. The agarose is poured into petri dishes or wells,allowed to solidify, and a test strain is applied to the agarose plate.The test strain is chosen, in part, on the intended application of theanalogue. Thus, by way of example, if an analogue with activity againstS. aureus is desired, an S. aureus strain is used. It may be desirableto assay the analogue on several strains and/or on clinical isolates ofthe test species. Plates are incubated overnight and, on the followingday, inspected visually for bacterial growth. The minimum inhibitoryconcentration (MIC) of an analogue is the lowest concentration ofpeptide that completely inhibits growth of the organism. Analogues thatexhibit good activity against the test strain, or group of strains,typically having an MIC of less than or equal to 16 μg/ml are selectedfor further testing.

[0091] The selected analogues may be further tested for their toxicityto normal mammalian cells. An exemplary assay is a red blood cell (RBC)(erythrocyte) hemolysis assay. Briefly, red blood cells are isolatedfrom whole blood, typically by centrifugation, and washed free of plasmacomponents. A 1% (v/v) suspension of erythrocytes in isotonic saline isincubated with different concentrations of peptide analogue. Generally,the analogue will be in a suitable formulation buffer. After incubationfor approximately 1 hour at 37° C., the cells are centrifuged, and theabsorbance of the supernatant at 540 nm is determined. A relativemeasure of lysis is determined by comparison to absorbance aftercomplete lysis of erythrocytes using NH₄Cl or equivalent (establishing a100% value). An analogue that is not lytic, or is only moderately lytic,as exemplified in Example 8, is desirable and is suitable for furtherscreening. Other in vitro toxicity assays, for example measurement oftoxicity towards cultured mammalian cells, may be used to assess invitro toxicity.

[0092] Solubility of the peptide analogue in formulation buffer is anadditional parameter that may be examined. Several different assays maybe used, such as appearance in buffer. Briefly, peptide analogue issuspended in solution, such as broth or formulation buffer. Theappearance is evaluated according to a scale that ranges from (a) clear,no precipitate, (b) light, diffuse precipitate, to (c) cloudy, heavyprecipitate. Finer gradations may be used. In general, less precipitateis more desirable. However, some precipitate may be acceptable.

[0093] Additional in vitro assays may be carried out to assess thepotential of the analogue as a therapeutic. Such assays include peptidesolubility in formulations, pharmacology in blood or plasma, serumprotein binding, analysis of secondary structure, for example bycircular dichroism, liposome permeabilization, and bacterial innermembrane permeabilization. In general, it is desirable that analoguesare soluble and perform better than indolicidin.

[0094] 2. In vivo Assays

[0095] Analogues selected on the basis of the results from the in vitroassays can be tested in vivo for efficacy, toxicity and the like.

[0096] The antibiotic activity of selected analogues may be assessed invivo for their ability to ameliorate microbial infections using animalmodels. Within these assays, an analogue is useful as a therapeutic ifinhibition of microorganismal growth compared to inhibition with vehiclealone is statistically significant. This measurement can be madedirectly from cultures isolated from body fluids or sites, orindirectly, by assessing survival rates of infected animals. Forassessment of antibacterial activity several animal models areavailable, such as acute infection models including those in which (a)normal mice receive a lethal dose of microorganisms, (b) neutropenicmice receive a lethal dose of microorganisms or (c) rabbits receive aninoculum in the heart, and chronic infection models. The model selectedwill depend in part on the intended clinical indication of the analogue.

[0097] By way of example, in one such normal mouse model, mice areinoculated ip or iv with a lethal dose of bacteria. Typically, the doseis such that 90-100% of animals die within 2 days. The choice of amicrorganismal strain for this assay depends, in part, upon the intendedapplication of the analogue, and in the accompanying examples, assaysare carried out with three different Staphylococclis strains. Briefly,shortly before or after inoculation (generally within 60 minutes),analogue in a suitable formulation buffer is injected. Multipleinjections of analogue may be administered. Animals are observed for upto 8 days post-infection and the survival of animals is recorded.Successful treatment either rescues animals from death or delays deathto a statistically significant level, as compared with non-treatmentcontrol animals. Analogues that show better efficacy than indolicidinitself are preferred.

[0098] In vivo toxicity of an analogue is measured throughadministration of a range of doses to animals, typically mice, by aroute defined in part by the intended clinical use. The survival of theanimals is recorded and LD₅₀, LD₉₀₋₁₀₀, and maximum tolerated dose (MTD)can be calculated to enable comparison of analogues. Analogues lesstoxic than indolicidin are preferred.

[0099] Additional in vivo assays may be performed to assist in theselection of analogues for clinical development. For example,immunogenicity of analogues can be evaluated, typically by injection ofthe analogue in formulation buffer into normal animals, generally mice,rats, or rabbits. At various times after injection, serum is obtainedand tested for the presence of antibodies that bind to the analogue.Testing after multiple injections, mimicking treatment protocols, mayalso be performed. Antibodies to analogues can be identified by ELISA,immunoprecipitation assays, Western blots, and other methods. (see,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988). Analogues that elicitno or minimal production of antibodies are preferred. Additionally,pharmacokinetics of the analogues in animals and histopathology ofanimals treated with analogues may be determined.

[0100] Selection of indolicidin analogues as potential therapeutics isbased on in vitro and in vivo assay results. In general, peptideanalogues that exhibit low toxicity at high dose levels and highefficacy at low dose levels are preferred candidates.

[0101] 3. Synergy Assays

[0102] For assessment of analogues in combination with an antibiotic oranother analogue, the combination can be subjected to the above seriesof assays. Antibiotics include any chemical that tends to prevent,inhibit or destroy life and as such, antibiotics include anti-bacterialagents, anti-fungicides, anti-viral agents, and anti-parasitic agents.Merely by way of example, anti-bacterial antibiotics are discussed.Methods for mixing and administering the components vary depending onthe intended clinical use of the combination.

[0103] Briefly, one assay for in vitro anti-bacterial activity, theagarose dilution assay, is set up with an array of plates that eachcontain a combination of peptide analogue and antibiotic in variousconcentrations. The plates are inoculated with bacterial isolates,incubated, and the MICs of the components recorded. These results arethen used to calculate the FIC. Antibiotics used in testing include, butare not limited to, penicillins, cephalosporins, carbacephems,cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides,quinolones, tetracyclines, macrolides, and fluoroquinolones (see Table 1below).

[0104] Examples of antibiotics, include, but are not limited to,Penicillin G (CAS Registry No.: 61-33-6); Methicillin (CAS Registry No.:61-32-5); Nafcillin (CAS Registry No.: 147-52-4); Oxacillin (CASRegistry No.: 66-79-5); Cloxacillin (CAS Registry No.: 61-72-3);Dicloxacillin (CAS Registry No.: 3116-76-5); Ampicillin (CAS RegistryNo.: 69-53-4); Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin(CAS Registry No.: 34787-01-4); Carbenicillin (CAS Registry No.:4697-36-3); Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin (CASRegistry No.: 37091-66-0); Piperacillin (CAS Registry No.: 61477-96-1);Imipenem (CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry No.:78110-38-0); Cephalothin (CAS Registry No.: 153-61-7); Cefazolin (CASRegistry No.: 25953-19-9); Cefaclor (CAS Registry No.: 70356-03-5);Cefamandole formate sodium (CAS Registry No.: 42540-40-9); Cefoxitin(CAS Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.:55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4); Cefmetazole (CASRegistry No.: 56796-20-4); Cefotetan (CAS Registry No.: 69712-56-7);Cefprozil (CAS Registry No.: 92665-29-7); Loracarbef (CAS Registry No.:121961-22-6); Cefetamet (CAS Registry No.: 65052-63-3); Cefoperazone(CAS Registry No.: 62893-19-0); Cefotaxime (CAS Registry No.:63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0); Ceftriaxone(CAS Registry No.: 73384-59-5); Ceftazidime (CAS Registry No.:72558-82-8); Cefepime (CAS Registry No.: 88040-23-7); Cefixime (CASRegistry No.: 79350-37-1); Cefpodoxime (CAS Registry No.: 80210-62-4);Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No.:79660-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin(CAS Registry No.: 70458-96-7); Ciprofloxacin (CAS Registry No.:85721-33-1); Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CASRegistry No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7);Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No.:564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CASRegistry No.: 60-54-8); Amikacin (CAS Registry No.: 37517-28-5);Gentamicin (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry No.:8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CASRegistry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1);Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CASRegistry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8);Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethylsuccinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate(CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS RegistryNo.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643-22-1);Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.:61036-64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin(CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.:738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin(CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1);Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS RegistryNo.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin(CAS Registry No.: 80214-83-1); Co-amoxiclavuanate; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives. TABLE 1 Class of Antibiotic Antibiotic Mode of ActionPENICILLINS Blocks the formation of new cell walls in bacteria NaturalPenicillin G, Benzylpenicillin Penicillin V, PhenoxymethylpenicillinPenicillinase resistant Methicillin, Nafcillin, Oxacillin Cloxacillin,Dicloxacillin Acylamino-penicillins Ampicillin, AmoxicillinCarboxy-penicillins Ticarcillin, Carbenicillin Ureido-penicillinsMezlocillin, Azlocillin, Piperacillin CARBAPENEMS Imipenem, MeropenemBlocks the formation of new cell walls in bacteria MONOBACTAMS Blocksthe formation of new cell walls in bacteria Aztreonam CEPHALOSPORINSPrevents formation of new cell walls in bacteria 1st GenerationCephalothin, Cefazolin 2nd Generation Cefaclor, Cefamandole Cefuroxime,Cefonicid, Cefmetazole, Cefotetan, Cefprozil 3rd Generation Cefetamet,Cefoperazone Cefotaxime, Ceftizoxime Ceftriaxone, Ceftazidime Cefixime,Cefpodoxime, Cefsulodin 4th Generation Cefepime CARBACEPHEMS LoracarbefPrevents formation of new cell walls in bacteria CEPHAMYCINS CefoxitinPrevents formation of new cell walls in bacteria QUINOLONES Fleroxacin,Nalidixic Acid Inhibits bacterial DNA Norfloxacin, Ciprofloxacinsynthesis Ofloxacin, Enoxacin Lomefloxacin, Cinoxacin TETRACYCLINESDoxycycline, Minocycline, Inhibits bacterial protein Tetracyclinesynthesis, binds to 30S ribosome subunit. AMINOGLYCOSIDES Amikacin,Gentamicin, Kanamycin, Inhibits bacterial protein Netilmicin,Tobramycin, synthesis, binds to 30S Streptomycin ribosome subunit.MACROLIDES Azitbromycin, Clarithromycin, inhibits bacterial proteinErythromycin synthesis, binds to 50S ribosome subunit Derivatives ofErythromycin estolate, Erythromycin Erythromycin stearate Erytbromycinethylsuccinate Erythromycin gluceptate Erythromycin lactobionateGLYCOPEPTIDES Vancomycin, Teicoplanin Inhibits cell wall synthesis,prevents peptidoglycan elongation. MISCELLANEOUS ChloramphenicolInhibits bacterial protein synthesis, binds to 50S ribosome subunit.Clindamycin Inhibits bacterial protein synthesis, binds to 50S ribosomesubunit. Trimethoprim Inhibits the enzyme dihydrofolate reductase, whichactivates folic acid. Sulfamethoxazole Acts as antimetabolite of PABA &inhibits synthesis of folic acid Nitrofurantoin Action unknown, but isconcentrated in urine where it can act on urinary tract bacteriaRifampin Inhibits bacterial RNA polymerase Mupirocin Inhibits bacteriallirotein synthesis

[0105] Synergy is calculated according to the formula below. An FIC of≦0.5 is evidence of synergy, although combinations with higher valuesmay be therapeutically useful.${\frac{{MIC}\left( {{peptide}\quad {in}\quad {combination}} \right)}{{MIC}\left( {{peptide}\quad {alone}} \right)} + \frac{{MIC}\left( {{antibiotic}\quad {in}\quad {combination}} \right)}{{MIC}\left( {{antibiotic}\quad {alone}} \right)}} = {FIC}$

[0106] For example, antibiotics from the groups of penicillins,cephalosporins, carbacephems, cephamycins, carbapenems, monobactams,aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides,fluoroquinolones, and other miscellaneous antibiotics may be used incombination with any of the peptides disclosed herein. For example, MBI11A1CN or MBI 11D18CN with Ciprofloxacin, MBI 11A1CN, MBI 11A3CN, MBI11B4CN, MBI 11D18CN or MBI 11G13CN with Mupirocin, MBI 11B9CN, MBI11D18CN or MBI 11F4CN with Piperacillin are preferred combinations.

[0107] C. Polymer Modification of Peptides and Proteins

[0108] As noted herein, the present invention provides methods andcompositions for modifying a compound with a free amine group, such aspeptides, proteins, certain antibiotics, and the like, with an activatedpolysorbate ester and derivatives. When the compounds are peptides orproteins, the modified or derivatized forms are referred to herein as“APS-modified peptides” or “APS-modified proteins”. Similarly, modifiedforms of antibiotics are referred to as “APS-modified antibiotics.”APS-modified compounds (e.g., APS-cationic peptides) have improvedpharmacological properties.

[0109] In addition to peptides and proteins, antibiotics, antifungals,anti-rythmic drugs, and any other compound with a free primary or otheramine are suitable for modification. For example, cephalosporins,aminopenicillins, ethambutol, pyrazinamide, sulfonamines, quinolones(e.g., ciprofiloxacin, clinafloxacin) aminoglycosides andspectinomhycins, including, but not limited to, streptomycin, neomycin,kanamycin, gentamicin, have free amines for modification. Anti-fungalssuch as amphotericin B, nystatin, 5-fluorocytosine, and the like haveamines available for derivativization. Anti-virals, such as tricyclicamines (e.g., amantadine); and anti-parasitic agents (e.g.,dapsone), mayall be derivatized. For exemplary purposes only, the discussion hereinis directed to modified peptides and proteins.

[0110] 1. Characteristics of Reagent

[0111] As discussed herein, a suitable reagent for formation ofAPS-modified compounds (e.g., peptides and proteins) comprises ahydrophobic region and a bydrophilic region, and optionally a linker.The hydrophobic region is a lipophilic compound with a suitablefunctional group for conjugation to the hydrophilic region or linker.The hydrophilic region is a polyalkylene glycol. As used herein,“polyalkylene glycol” refers to 2 or 3 carbon polymers of glycols. Twocarbon polyalkylenes include polyethylene glycol (PEG) of variousmolecular weights, and its derivatives, such as polysorbate. Threecarbon polyalkylenes include polypropylene glycol and its derivatives.

[0112] The hydrophobic region is generally a fatty acid, but may be afatty alcohol, fatty thiol, and the like, which are also lipophiliccompounds. The fatty acid may be saturated or unsaturated. The chainlength does not appear to be important, although typically commerciallyavailable fatty acids are used and have chain lengths of C₁₂₋₁₈. Thelength may be limited however by solubility or solidity of the compound,that is longer lengths of fatty acids are solid at room temperature.Fatty acids of 12 carbons (lauryl), 14 carbons, 16 carbons (palmitate),and 18 carbons (monostearate or oleate) are preferred chain lengths.

[0113] The hydrophilic region is a polyalkylene glycol, eitherpolyethylene or polypropylene glycol monoether. The ether function isformed by the linkage between the polyoxyethylene chain, preferablyhaving a chain length of from 2 to 100 monomeric units, and the sorbitangroup. Polymethylene glycol is unsuitable for administration in animalsdue to formation of formaldehydes, and glycols with a chain length of ≧4may be insoluble. Mixed polyoxyethylene-polyoxypropylene chains are alsosuitable.

[0114] A linker for bridging the hydrophilic and hydrophobic regions isnot required, but if used, should be a bifunctional nucleophile able toreact with both polyalkylene glycol and the hydrophobic region. Thelinker provides electrons for a nucleophilic reaction with thepolyalkylene glycol, typically formed by reaction with ethylene oxide orpropylene oxide. Suitable linkers include sorbitan, sugar alcohols,ethanolamine, ethanolthiol, 2-mercaptoethanol, 1,6 diaminohexane, anamino acid (e.g., glutamine, lysine), other reduced sugars, and thelike. For example, sorbitan forms an ester linkage with the fatty acidin a polysorbate.

[0115] Suitable compounds include polyoxyethylenesorbitans, such as themonolaurate, monooleate, monopalmitate, monostearate, trioleate, andtristearate esters. These and other suitable compounds may besynthesized by standard chemical methods or obtained commercially (e.g.,Sigma Chemical Co., MO; Aldrich Chemical Co., WI; J. B. Baker, NJ).

[0116] 2. Activation of Reagent

[0117] The reagent, generally a polysorbate, is activated by exposure toUV light with free exchange of air. Activation is achieved using a lampthat irradiates at 254 nm or 302 nm. Preferably, the output is centeredat 254 nm. Longer wave lengths may require longer activation time. Whilesome evidence exists that fluorescent room light can activate thepolysorbates, experiments have shown that use of UV light at 254 nmyields maximal activation before room light yields a detectable level ofactivation.

[0118] Air plays an important role in the activation of thepolysorbates. Access to air doubles the rate of activation relative toactivations performed in sealed containers. It is not yet known whichgas is responsible; an oxygen derivative is likely, although peroxidesare not involved. UV exposure of compounds with ether linkages is knownto generate peroxides, which can be detected and quantified usingperoxide test strips. In a reaction, hydrogen peroxide at 1 to 10 foldhigher level than found in UV-activated material was added to apolysorbate solution in the absence of light. No activation wasobtained.

[0119] The reagent is placed in a suitable vessel for irradiation. Aconsideration for the vessel is the ability to achieve uniformirradiation. Thus, if the pathlength is long, the reagent may be mixedor agitated. The activation requires air; peroxides are not involved inthe activation. The reagent can be activated in any aqueous solution andbuffering is not required.

[0120] An exemplary activation takes place in a cuvette with a 1 cmliquid thickness. The reagent is irradiated at a distance of less than 9cm at 1500 μW/cm² (initial source output) for approximately 24 hours.Under these conditions, the activated reagent converts a minimum of 85%of the peptide to APS-peptide.

[0121] 3. Modification of Peptides or Proteins with Activated Reagent

[0122] The peptides or proteins are reacted with the APS reagent ineither a liquid or solid phase and become modified by the attachment ofthe APS derivative. The methods described herein for attachment offerthe advantage of maintaining the charge on the peptide or protein. Whenthe charge of the peptide is critical to its function, such as theantibiotic activity of cationic peptides described herein, theseattachment methods offer additional advantages. Methods that attachgroups via acylation result in the loss of positive charge viaconversion of amino to amido groups. In addition, no bulky orpotentially antigenic linker, such as a triazine group, is known to beintroduced by the methods described herein.

[0123] As noted above, APS-peptide formation occurs in solid phase or inaqueous solution. Briefly, in the solid phase method, the peptide issuspended in a suitable buffer, such as an acetate buffer. Othersuitable buffers that support APS-peptide formation may also be used.The acetate buffer may be sodium, rubidium, lithium, and the like. Otheracetate solutions, such as HAc or HAc-NaOH, are also suitable. Apreferred pH range for the buffer is from 2 to 8.3, although a widerrange may be used. When the starting pH of the acetic acid-NaOH bufferis varied, subsequent lyophilization from 200 mM acetic acid bufferyields only the Type I modified peptide (see Example 14). The presenceof an alkaline buffer component results in the formation of Type IImodified peptides. A typical peptide concentration is 1 mg/ml, whichresults in 85-95% modified peptide, however other concentrations aresuitable. The major consideration for determining concentration appearsto be economic. The activated polymer (APS) is added in molar excess tothe peptide, such that a 1:1 molar ratio of APS-modified peptide isgenerated. Generally, a starting ratio of approximately 2.5:1(APS:peptide) to 5:1 (APS: peptide) yields a 1:1 APS-modified peptide.

[0124] The reaction mix is then frozen (e.g., −80° C.) and lyophilized.Sodium acetate disproportionates into acetic acid and NaOH duringlyophilization; removal of the volatile acetic acid by the vacuum leavesNaOH dispersed throughout the result solid matrix. This loss of aceticacid is confirmed by a pH increase detected upon dissolution of thelyophilizate. No APS-modified peptide is formed in acetate buffer if thesamples are only frozen then thawed.

[0125] The modification reaction can also take place in aqueoussolution. However, APS modifications do not occur at ambient temperaturein any acetate buffer system tested regardless of pH. APS modificationsalso are not formed in phosphate buffers as high as pH 11.5. APSmodification does occur in a sodium carbonate buffer at a pH greaterthan about 8.5. Other buffers may also be used if they supportderivitization. A pH range of 9-11 is also suitable, and pH 10 is mostcommonly used. The reaction occurs in two phases: Type I peptides formfirst, followed by formation of Type II peptides.

[0126] In the present invention, linkage occurs at an amino group. For apeptide, linkage can occur at the α-NH₂ of the N-terminal amino acid orε-NH₂ group of lysine. Other primary and secondary amines may also bemodified. Complete blocking of all amino groups by acylation (MBI11CN-Y1) inhibits APS-peptide formation. Thus, modification of arginineor tryptophan residues does not occur. If the only amino group availableis the α-amino group (e.g., MBI 11B9CN and MBI 11G14CN), the Type I formis observed. The inclusion of a single lysine (e.g., MBI 11B1CN, MBI11B7CN, MBI 11B8CN), providing an ε-amino group, results in Type IIforms as well. The amount of Type II formed increases for peptides withmore lysine residues.

[0127] 4. Purification and Physical Properties of APS-Modified Peptides

[0128] The APS-modified peptides may be purified. In circumstances inwhich the free peptide is toxic, purification may be necessary to removeunmodified peptide and/or unreacted polysorbate. Any of a variety ofpurification methods may be used. Such methods include reversed phaseHPLC, precipitation by organic solvent to remove polysorbate, sizeexclusion chromatography, ion exchange chromatography, filtration andthe like. RP-HPLC is preferred. Procedures for these separation methodsare well known.

[0129] APS-peptide (or protein) formation results in the generation ofpeptide-containing products that are more hydrophobic that the parentpeptide. This property can be exploited to effect separation of theconjugate from free peptide by RP-HPLC. The conjugates are resolved intotwo populations based on their hydrophobicity as determined by RP-HPLC;the Type I population elutes slightly earlier than the Type IIpopulation.

[0130] The MBI 11 series of peptides have molecular weights between 1600and 2500. When run on a Superose 12 column, a size exclusion column,these peptides elute no earlier than the bed volume indicating amolecular mass below 20 kDa. In contrast, the APS-modified peptideselute at 50 kDa, thus demonstrating a large increase in apparentmolecular mass.

[0131] An increase in apparent molecular mass could enhance thepharmacokinetics of the cationic peptides because increased molecularmass reduces the rate at which peptides and proteins are removed fromblood. Micelle formation may offer additional benefits by delivering“packets” of peptide molecules to microorganisms rather than relying onthe multiple binding of single peptide molecules. In addition, theAPS-modified peptides are soluble in methylene chloride or chloroform,whereas the parent peptide is essentially insoluble. This increasedorganic solubility may significantly enhance the ability to penetratetissue barriers.

[0132] In addition, by circular dichroism (CD) studies, APS-modifiedpeptides are observed to have an altered 3-dimensional conformation. Asshown in the Examples, MBI 11CN and MBI 11B7CN have unordered structuresin phosphate buffer or 40% aqueous trifluoroethanol (TFE) and form aβ-turn conformation only upon insertion into liposomes. In contrast, CDspectra for APS-modified MBI 11CN and APS-modified MBI 11B7CN indicateβ-turn structure in phosphate buffer.

[0133] D. Formulations and Administration

[0134] As noted above, the present invention provides methods fortreating and preventing infections by administering to a patient atherapeutically effective amount of a peptide analogue of indolicidin asdescribed herein. Patients suitable for such treatment may be identifiedby well-established hallmarks of an infection, such as fever, pus,culture of organisms, and the like. Infections that may be treated withpeptide analogues include those caused by or due to microorganisms.Examples of microorganisms include bacteria (e.g., Gram-positive,Gram-negative), fungi, (e.g., yeast and molds), parasites (e.g.,protozoans, nematodes, cestodes and trematodes), viruses, and prions.Specific organisms in these classes are well known (see for example,Davis et al., Microbiology, 3^(rd) edition, Harper & Row, 1980).Infections include, but are not limited to, toxic shock syndrome,diphtheria, cholera, typhus, meningitis, whooping cough, botulism,tetanus, pyogenic infections, dysentery, gastroenteritis, anthrax, Lymedisease, syphilis, rubella, septicemia and plague.

[0135] Effective treatment of infection may be examined in severaldifferent ways. The patient may exhibit reduced fever, reduced number oforganisms, lower level of inflammatory molecules (e.g., IFN-γ, IL-12,IL-1, TNF), and the like.

[0136] Peptide analogues of the present invention are preferablyadministered as a pharmaceutical composition. Briefly, pharmaceuticalcompositions of the present invention may comprise one or more of thepeptide analogues described herein, in combination with one or morephysiologically acceptable carriers, diluents, or excipients. As notedherein, the formulation buffer used may affect the efficacy or activityof the peptide analogue. A suitable formulation buffer contains bufferand solubilizer. The formulation buffer may comprise buffers such assodium acetate, sodium citrate, neutral buffered saline,phosphate-buffered saline, and the like or salts, such as NaCl. Sodiumacetate is preferred. In general, an acetate buffer from 5 to 500 mM isused, and preferably from 100 to 200 mM. The pH of the final formulationmay range from 3 to 10, and is preferably approximately neutral (aboutpH 7-8). Solubilizers, such as polyoxyethylenesorbitans (e.g., Tween 80,Tween 20) and polyoxyethylene ethers (e.g., Brij 56) may also be addedif the compound is not already APS-modified.

[0137] Although the formulation buffer is exemplified herein withpeptide analogues of the present invention, this buffer is generallyuseful and desirable for delivery of other peptides. Peptides that maybe delivered in this formulation buffer include indolicidin, otherindolicidin analogues (see, PCT WO 95/22338), bacteriocins, gramicidin,bactenecin, drosocin, polyphemusins, defensins, cecropins, melittins,cecropin/melittin hybrids, magainins, sapecins, apidaecins, protegrins,tachyplesins, thionins; IL-1 through 15; corticotropin-releasinghormone; human growth hormone; insulin; erythropoietin; thrombopoietin;myelin basic protein peptides; various colony stimulating factors suchas M-CSF, GM-CSF, kit ligand; and peptides and analogues of these andsimilar proteins.

[0138] Additional compounds may be included in the compositions. Theseinclude, for example, carbohydrates such as glucose, mannose, sucrose ordextrose, mannitol, other proteins, polypeptides or amino acids,chelating agents such as EDTA or glutathione, adjuvants andpreservatives. As noted herein, pharmaceutical compositions of thepresent invention may also contain one or more additional activeingredients, such as an antibiotic (see discussion herein on synergy) orcytokine.

[0139] The compositions may be administered in a delivery vehicle. Forexample, the composition can be encapsulated in a liposome (see, e.g.,WO 96/10585; WO 95/35094), complexed with lipids, encapsulated inslow-release or sustained release vehicles, such as poly-galactide, andthe like. Within other embodiments, compositions may be prepared as alyophilizate, utilizing appropriate excipients to provide stability.

[0140] Pharmaceutical compositions of the present invention may beadministered in various manners. For example, peptide analogues may beadministered by intravenous injection, intraperitoneal injection orimplantation, subcutaneous injection or implantation, intradermalinjection, lavage, inhalation, implantation, intramuscular injection orimplantation, intrathecal injection, bladder wash-out, suppositories,pessaries, topical (e.g., creams, ointments, skin patches, eye drops,ear drops, shampoos) application, enteric, oral, or nasal route. Theanalogue may be applied locally as an injection, drops, spray, tablets,cream, ointment, gel, and the like. Analogue may be administered as abolus or as multiple doses over a period of time.

[0141] The level of peptide in serum and other tissues afteradministration can be monitored by various well-established techniquessuch as bacterial, chromatographic or antibody based, such as ELISA,assays.

[0142] Pharmaceutical compositions of the present invention areadministered in a manner appropriate to the infection or disease to betreated. The amount and frequency of administration will be determinedby factors such as the condition of the patient, the cause of theinfection, and the severity of the infection. Appropriate dosages may bedetermined by clinical trials, but will generally range from about 0.1to 50 mg/kg.

[0143] In addition, the analogues of the present invention may be usedin the manner of common disinfectants or in any situation in whichmicroorganisms are undesirable. For example, these peptides may be usedas surface disinfectants, coatings, including covalent bonding, formedical devices, coatings for clothing, such as to inhibit growth ofbacteria or repel mosquitoes, in filters for air purification, such ason an airplane, in water purification, constituents of shampoos andsoaps, food preservatives, cosmetic preservatives, media preservatives,herbicide or insecticides, constituents of building materials, such asin silicone sealant, and in animal product processing, such as curing ofanimal hides. As used herein, “medical device” refers to any device foruse in a patient, such as an implant or prosthesis. Such devicesinclude, stents, tubing, probes, cannulas, catheters, synthetic vasculargrafts, blood monitoring devices, artificial heart valves, needles, andthe like.

[0144] For these purposes, typically the peptides alone or inconjunction with an antibiotic are included in compositions commonlyemployed or in a suitable applicator, such as for applying to clothing.They may be incorporated or impregnated into the material duringmanufacture, such as for an air filter, or otherwise applied to devices.The peptides and antibiotics need only be suspended in a solutionappropriate for the device or article. Polymers are one type of carrierthat can be used.

[0145] The analogues, especially the labeled analogues, may be used inimage analysis and diagnostic assays or for targeting sites ineukaryotic multicellular and single cell cellular organisms and inprokaryotes. As a targeting system, the analogues may be coupled withother peptides, proteins, nucleic acids, antibodies and the like.

[0146] The following examples are offered by way of illustration, andnot by way of limitation.

EXAMPLES Example 1 Synthesis Purification and Characterization ofPeptide Analogues

[0147] Peptide synthesis is based on the standard solid-phase Fmocprotection strategy. The instrument employed is a 9050 PlusPepSynthesiser (PerSeptive BioSystems Inc.). Polyethylene glycolpolystyrene (PEG-PS) graft resins are employed as the solid phase,derivatized with an Fmoc-protected amino acid linker for C-terminalamide synthesis. HATU(O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) is used as the coupling reagent. During synthesis,coupling steps are continuously monitored to ensure that each amino acidis incorporated in high yield. The peptide is cleaved from thesolid-phase resin using trifluoroacetic acid and appropriate scavengersand the crude peptide is purified using preparative reversed-phasechromatography.

[0148] All peptides are analyzed by mass spectrometry to ensure that theproduct has the expected molecular mass. The product should have asingle peak accounting for >95% of the total peak area when subjected toanalytical reversed-phase high performance liquid chromatography(RP-HPLC). In addition, the peptide should show a single band accountingfor >90% of the total band intensity when subjected to acid-urea gelelectrophoresis.

[0149] Peptide content, the amount of the product that is peptide ratherthan retained water, salt or solvent, is measured by quantitative aminoacid analysis, free amine derivatization or spectrophotometricquantitation. Amino acid analysis also provides information on the ratioof amino acids present in the peptide, which assists in confirming theauthenticity of the peptide.

[0150] Peptide analogues and their names are listed in Table 2. In thistable, and elsewhere, the amino acids are denoted by the one-letteramino acid code and lower case letters represent the D-form of the aminoacid. TABLE 2 10 I L P W K W P W W P W R R 10CN I L P W K W P W W P W RR 11 I L K K W P W W P W R R K 11CN I L K K W P W W P W R R K 11CNR K RR W P W W P W K K L I 11A1CN J L K K F P F F P F R R K 11A2CN I L K K IP I I P I R R K 11A3CN I L K K Y P Y Y P Y R R K 11A4CN I L K K W P W PW R R K 11A5CN I L K K Y P W Y P W R R K 11A6CN I L K K F P W F P W R RK 11A7CN I L K K F P F W P W R R K 11ABCN I L R Y V Y Y V Y R R K 11B1CNI L R R W P W W P W R R K 11B2CN I L R R W P W W P W R K 11B3CN I L K WP W W P W R R K 11B4CN I L K K W P W W P W R K 11B5CN I L K W P W W P WR K 11B7CN I L R W P W W P W R R K 11B7CNR K R R W P W W P W R L I11B8CN I L W P W W P W R R K 11B9CN I L R R W P W W P W R R R 11B10CN IL K K W P W W P W K K K 11B16CN I L R W P W W P W R R K I M I L K K A GS 11B17CN I L R W P W W P W R R K M I L K K A G S 11B18CN I L R W P W WP W R R K D M I L K K A G S 11C3CN I L K K W A W W P W R R K 11C4CN I LK K W P W W A W R R K 11C5CN W W K K W P W W P W R R K 11D1CN L K K W PW W P W R R K 11D3CN P W W P W R R K 11D4CN I L K K W P W W P W R R K MI L K K A G S 11D5CN I L K K W P W W P W R R M I L K K A G S 11D6CN I LK K W P W W P W R R I M I L K K A G S 11D11H I L K K W P W W P W R R K M11D12H I L K K W P W W P W R R M 11D13H I L K K W P W W P W R R I M11D14CN I L K K W W W P W R K 11D15CN I L K K W P W W W R K 11D18CN W RI W K P K W R L P K W 11E1CN I L K K W P W W P W R R K 11E2CN I L K K WP W W P W R R k 11E3CN I L K K W P W W P W R R k 11F1CN I L K K W V W WV W R R K 11F2CN I L K K W P W W V W R R K 11F3CN I L K K W V W W P W RR K 11F4CN I L R W V W W V W R R K 11F4CNR K R R W V W W V W R L I11G2CN I K K W P W W P W R R K 11G3CN I L K K P W W F W R R K 11G4CN I LK K W W W P W R R K 11G5CN I L K K W P W W W R R K 11G6CN I L K K W P WW P R R K 11G7CN I L K K W P W W P W R R 11G13CN I L K K W P W W P W K11G14CN I L K K W P W W P W R 11H1CN A L R W P W W P W R P K 11H2CN I AR W P W W P W R R K 11H3CN I L A W P W W P W R R K 11H4CN I L R A P W WP W R R K 11H5CN I L R W A W W P W R R K 11H6CN I L R W P A W P W R R K11H7CN I L R W P W A P W R R K 11H8CN I L R W P W W A W R R K 11H9CN I LR W P W W P A R R K 11H10CN I L R W P W W P W A R K 11H11CN I L R W P WW P W R A K 11H12CN I L R W P W W P W R R A

Example 2 Synthesis of Modified Peptides

[0151] Indolicidin analogues are modified to alter the physicalproperties of the original peptide. Such modifications include:acetylation at the N-terminus, Fmoc-derivatized N-terminus,polymethylation, peracetylation, and branched derivatives.

[0152] α-N-terminal acetylation. Prior to cleaving the peptide from theresin and deptrotecting it, the fully protected peptide is treated withN-acetylimidazole in DMF for 1 hour at room temperature, which resultsin selective reaction at the α-N-terminus. The peptide is thendeprotected/cleaved and purified as for an unmodified peptide.

[0153] Fmoc-derivatized α-N-terminus. If the final Fmoc deprotectionstep is not carried, the α-N-terminus Fmoc group remains on the peptide.The peptide is then side-chain deprotected/cleaved and purified as foran unmodified peptide.

[0154] Polymethylation. The purified peptide in a methanol solution istreated with excess sodium bicarbonate, followed by excess methyliodide. The reaction mixture is stirred overnight at room temperature,extracted with organic solvent, neutralized and purified as for anunmodified peptide. Using this procedure, a peptide is not fullymethylated; methylation of MBI 11CN yielded an average of 6 methylgroups. Thus, the modified peptide is a mixture of methylated products.

[0155] Peracetylation. A purified peptide in DMF solution is treatedwith N-acetylimidazole for 1 hour at room temperature. The crude productis concentrated, dissolved in water, lyophilized, re-dissolved in waterand purified as for an unmodified peptide. Complete acetylation ofprimary amine groups is observed.

[0156] Four/eight branch derivatives. The branched peptides aresynthesized on a four or eight branched core bound to the resin.Synthesis and deprotection/cleavage proceed as for an unmodifiedpeptide. These peptides are purified by dialysis against 4 M guanidinehydrochloride then water, and analyzed by mass spectrometry.

[0157] Peptides modified using the above procedures are listed in Table3. TABLE 3 Peptide Peptide modified name Sequence Modification 10 10A IL P W K W P W W P W R R Acetylated α-N-terminus 11 11 I L K K W P W W PW R R K Acetylated α-N-terminus 11CN 11CAN I L K K W P W W P W R R KAcetylated α-N-terminus 11CN 11CNW1 I L K K W P W W P W R R KFmoc-derivatized N-terminus 11CN 11CNX1 I L K K W P W W P W R R KPolymethylated derivative 11CN 11CNY1 I L K K W P W W P W R P KPeracetylated derivative 11 11M4 I L K K W P W W P W R P K Four branchderivative 11 11M8 I L K K W P W W P W R R K Eight branch derivative11B1CN 11B1GNW1 I L R R W P W W P W R P K Fmoc-derivatized N-terminus11B4CN 11B4ACN I L K K W P W W P W R K Acetylated N-terminus 11B9CN11B9ACN I L P R W P W W P W R R R Acetylated N-terminus 11D9 11D9M8 W WP W R R K Eight branch derivative 11D10 11D10M8 I L K K W P W Eightbranch derivative 11G6CN 11G6ACN I L K K W P W W P P P K Acetylatedα-N-terminus 11G7CN 11G7ACN I L K K W P W W P W P P Acetylatedα-N-terminus

Example 3 Recombinant Production of Peptide Analogues

[0158] Peptide analogues are alternatively produced by recombinant DNAtechnique in bacterial host cells. The peptide is produced as a fusionprotein, chosen to assist in transporting the fusion peptide toinclusion bodies, periplasm, outer membrane or extracellularenvironment.

[0159] Construction of Plasmids Encoding MBI-11 Peptide Fusion Protein

[0160] Amplification by polymerase chain reaction is used to synthesizedouble-stranded DNA encoding the MBI peptide genes from single-strandedtemplates. For MBI-11, 100 μl of reaction mix is prepared containing 50to 100 ng of template, 25 pmole of each primer, 1.5 mM MgCl₂, 200 μM ofeach dNTP, 2U of Taq polymerase in the supplier's buffer. The reactionsproceeded with 25 cycles of 94° C. for 30 sec., 55° C. for 30 sec., 74°C. for 30 sec., followed by 74° C. for 1 min. Amplified product isdigested with BamHI and HindIII and cloned into a plasmid expressionvector encoding the fusion partner and a suitable selection marker.

[0161] Production of MBI-11 Peptide Fusion in E. coli

[0162] The plasmid pR2h-11, employing a T7 promoter, high copy origin ofreplication, Ap^(r) marker and containing the gene of the fusionprotein, is co-electroporated with pGP1-2 into E. coli strain XL1-Blue.Plasmid pGP1-2 contains a T7 RNA polymerase gene under control of alambda promoter and cI857 repressor gene. Fusion protein expression isinduced by a temperature shift from 30° C. to 42° C. Inclusion bodiesare washed with solution containing solubilizer and extracted withorganic extraction solvent. Profiles of the samples are analyzed bySDS-PAGE. FIG. 1 shows the SDS-PAGE analysis and an extraction profileof inclusion body from whole cell. The major contaminant in the organicsolvent extracted material is β-lactamase (FIG. 1). The expression levelin these cells is presented in Table 4. TABLE 4 % which is FusionMol.mass % protein in % in inclusion MBI-11 protein (kDa) whole celllysate body extract peptide MBI-11 20.1 15 42 7.2

[0163] In addition, a low-copy-number vector, pPD100, which contains achloramphenicol resistance gene, is used to express MBI-11 in order toeliminate the need for using ampicillin, thereby reducing the appearanceof β-lactamase in extracted material. This plasmid allows selective geneexpression and high-level protein overproduction in E. coli using thebacteriophage T7 RNA polymerase/T7 promoter system (Dersch et al., FEMSMicrobiol. Lett. 123: 19-26,1994). pPD100 contains a chloramphenicolresistance gene (CAT) as a selective marker, a multiple cloning site,and an ori sequence derived from the low-copy-number vector pSC101.There are only about 4 to 6 copies of these plasmids per host cell. Theresulting construct containing MBI-11 is called pPDR2h-11. FIG. 2presents a gel electrophoresis analysis of the MBI-11 fusion proteinexpressed in this vector. Expression level of MBI-11 fusion protein iscomparable with that obtained from plasmid pR2h-11. The CAT gene productis not apparent, presumably due to the low-copy-number nature of thisplasmid, CAT protein is not expressed at high levels in pPDR2h-11.

Example 4 In vitro Assays to Measure Peptide Analogue Activity

[0164] Agarose Dilution Assay

[0165] The agarose dilution assay measures antimicrobial activity ofpeptides and peptide analogues, which is expressed as the minimuminhibitory concentration (MIC) of the peptides.

[0166] In order to mimic in vivo conditions, calcium and magnesiumsupplemented Mueller Hinton broth is used in combination with a low EEOagarose as the bacterial growth medium. The more commonly used agar isreplaced with agarose as the charged groups in agar prevent peptidediffusion through the media. The media is autoclaved and then cooled to50-55° C. in a water bath before aseptic addition of antimicrobialsolutions. The same volume of different concentrations of peptidesolution are added to the cooled molten agarose that is then poured to adepth of 3-4 mm.

[0167] The bacterial inoculum is adjusted to a 0.5 McFarland turbiditystandard (PML Microbiological) and then diluted 1:10 before applicationon to the agarose plate. The final inoculum applied to the agarose isapproximately 10⁴ CFU in a 5-8 mm diameter spot. The agarose plates areincubated at 35-37° C. for 16 to 20 hours.

[0168] The MIC is recorded as the lowest concentration of peptide thatcompletely inhibits growth of the organism as determined by visualinspection. Representative MICs for various indolicidin analogues areshown in the Table 5 below. TABLE 5 Organism Organism # MIC (μg/ml) 1.MBI 10 A. calcoaceticus AC001 128 E. coli ECO002 128 E. faecalis EFS0048 K. pneumoniae KP001 128 P. aeruginosa PA003 >128 S. aureus SA007 2 S.maltophilia SMA001 128 S. marcescens SMS003 >128 2. MBI 10A E. faecalisEFS004 16 E. faecium EFM003 8 S. aureus SA010 8 3. MBI 10CN A.calcoaceticus AC001 64 E. cloacae ECL007 >128 E. coli ECO001 32 E. coliSBECO2 16 E. faecalis EFS004 8 E. faecium EFM003 2 K. pneumoniae KP00264 P. aeruginosa PA002 >128 S. aureus SA003 2 S. epiderimidis SE010 4 S.maltophilia SMA002 64 S. marcescens SMS004 >128 4. MBI 11 A.calcoaceticus AC002 8 E. cloacae ECL007 >128 E. coli ECO002 64 E.faecium EFM003 4 E. faecalis EFS002 64 K. pneumoniae KP001 128 P.aeruginosa PA004 >128 S. aureus SA004 4 S. maltophilia SMA002 128 S.marcescens SMS004 >128 5. MBI 11A A. calcoaceticus AC001 >64 E. cloacaeECL007 >64 E. coli ECO005 >64 E. faecalis EFS004 32 K. pneumoniae KP00164 P. aeruginosa PA024 >64 S. aurdus SA002 4 S. maltophilia SMA002 >64S. marcescens SMS003 >64 6. MBI 11ACN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 16 E. faccalis EFS004 8 E. faecalisEFS008 64 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S. aureusSA014 8 S. epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescensSMS003 >128 7. MBI 11CN A. calcoaceticus AC001 128 E. cloacae ECL007 >64E. coli ECO002 8 E. faecium EFM001 8 E. faecalis EFS001 32 H. influenzaeHIN001 >128 K. pneumoniae KP002 128 P. aeruginosa PA003 >128 P.mirabilis PM002 >128 S. aureus SA003 2 S. marcescens SBSM1 >128 S.pneumoniae SBSPN2 >128 S. epidermidis SE001 2 S. maltophilia SMA001 64S. marcescens SMS003 >128 S. pyogenes SPY003 8 8. MBI 11CNR A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 8 E.faecalis EFS001 4 K. pneumoniae KP001 4 P. aeruginosa PA004 32 S. aureusSA093 4 S. epidermidis SE010 4 S. maltophilia SMA002 32 S. marcescensSMS003 128 9. MBI 11CNW1 A. calcoaceticus AC002 8 E. cloacae ECL007 64E. coli ECO005 32 E. faecalis EFS00I 8 K. pneumoniae IQP00I 32 P.aeruginosa PA004 64 S. aureus SA010 4 S. maltophilia SMA002 32 S.marcescens SMS003 >128 10. MBI 11CNX1 A. calcoaceticus AC001 >64 E.cloacac ECL007 >64 E. coli ECO005 64 E. faecalis EFS004 16 K. pneumoniaeKIP001 >64 P. aeruginosa PA024 >64 S. aureus SA006 2 S. maltophiliaSMA002 >64 S. marcescens SMS003 >64 11. MBI 11CNY1 A. calcoaceticusAC001 >64 E. cloacac ECL007 >64 E. coli ECO005 >64 E. faecalisEFS004 >64 K. pneumnoniae KP001 >64 P. aeruginosa PA004 >64 S. aureusSA006 16 S. epidermidis SE010 128 S. maltophilia SMA002 >64 S.marcescens SMS003 >64 12. MBI 11M4 E. faecium EFM001 32 E. faecalisEFS001 32 S. aureus SA008 8 13. MBI 11M8 E. faecalis EFS002 32 E.faecium EFM002 32 S. aureus SA008 32 14. MBI 11A1CN A. calcoaceticusAC002 16 E. cloacae ECL007 >128 E. coli ECO002 32 E. faecium EFM002 1 E.faecalis EFS002 32 H. influenzae HIN002 >128 K. pneumoniae KP002 >128 P.aeruginosa PA004 >128 S. aureus SA005 8 P. vulgaris SBPV1 >128 S.marcescens SBSM2 >128 S. pneumoniae SBSPN2 >128 S. epidermidis SE002 16S. maltophilia SMA002 >128 15. MBI 11A2CN A. calcoaceticus AC001 >128 E.cloacae ECL007 >128 E. coli ECO003 >128 E. faecium EFM003 16 E. faecalisEFS002 >128 K. pneumoniae KP002 >128 P. aeruginosa PA004 >128 S. aureusSA004 8 S. maltophilia SMA001 >128 S. marcescens SMS003 >128 16. MBI11A3CN A. calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coliECO002 >128 E. faecium EFM003 64 E. feacalis EFS002 >128 H. influenzaeHIN002 >128 K. pneumoniae KP001 >128 P. aeruginosa PA002 >128 S. aureusSA004 32 P. vulgaris SBPV1 >128 S. marcescens SBSM2 >128 S. pneumoniaeSBSPN3 >128 S. epidermidis SE002 128 S. maltophilia SMA001 >128 17. MBI11A4CN A. calcoaceticus AC002 8 E. cloacae ECL007 >128 E. coli ECO003 32E. faecalis EFS002 64 E. faecium EFM001 32 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA005 2 S. epidermidis SE002 8 S.maltophilia SMA002 >128 S. marcescens SMS004 >128 18. MBI 11A5CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli. ECO003 128 E.faecium EFM003 4 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA003 >128 S. aureus SA002 16 S. maltophilia SMA002 >128 S.marcescens SMS003 >128 19. MBI 11A6CN E. faecium EFM003 2 E. aecalisEFS004 64 S. aureus SA016 2 20. MBI 11A7CN E. faecium EFM003 2 E.faecalis EFS002 16 S. aureus SA009 2 21. MBI 11A8CN A. calcoaceticusAC002 8 E. cloacae ECL007 >128 E. coli ECO005 32 E. faecalis BES001 4 K.pneumoniae KP001 128 P. aeruginosa PA004 >128 S. aureus SA093 1 S.epidermidis SE010 16 S. maltophilia SMA002 32 S. marcescens SMS003 >12822. MBI 11B1CN A. calcoaceticus AC001 32 E. cloacac ECL007 >128 E. coliECO003 8 E. faecium EFM002 2 E. faecalis EFS004 8 K. pneumoniae KP002 64P. aeruginosa PA005 >128 S. aureus SA005 2 S. epidermiclis SE001 2 S.malto philia SMA001 64 S. marcescens SMS004 >128 23. MBI 11B1CNW1 A.calcoaceticus AC002 16 E. cloacae ECL007 64 E. coli ECO005 32 E.faecalis EFS004 8 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S.aureus SA014 16 S. epidermidis SE010 8 S. maltophilia SMA002 32 S.marcescens SMS003 >128 24. MBI 11B2CN A. calcoaceticus AC001 64 E.cloacac ECL007 >128 E. coli ECO003 16 E. faecium EFM001 8 E. faecalisEFS004 8 K. pneumoniae KP002 64 P. aeruginosa PA003 >128 S. aureus SA0052 S. maltophilia SMA002 64 S. marcescens SMS004 >128 25. MBI 11B3CN A.calcoaceticus AC001 64 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 8 E. faecalis EFS001 16 K. pneumoniae KP002 64 P.aeruginosa PA003 >128 S. aureus SA010 4 S. maltophilia SMA002 32 S.marcescens SMS004 >128 26. MBI 11B4CN A. calcoaceticus AC001 >128 E.cloacac ECL007 >128 E. coli ECO003 16 E. faecalis EFS002 16 H.influenzae HIN002 >128 K. pneumoniae KP002 128 P. aeruginosa PA006 >128S. aureus SA004 2 S. marcescens SBSM2 >128 S. pneumoniae SBSPN3 128 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS004 >12827. MBI 11B4ACN A. calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coliECO005 32 E. faecalis EFS008 64 K. pneumoniae KP001 32 P. aeruginosaPA004 >128 S. aureus SA008 1 S. epiderimidis SF010 8 S. maltophiliaSMA002 64 S. marcescens SMS003 >128 28. MBI 11B5CN E. faecium EFM002 1E. faecalis EFS002 16 S. aureus SA005 2 29. MBI 11B7 A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS008 8 K.pneumoniae KP001 16 P. aeruginosa PA004 >128 S. aureus SA093 1 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS003 >12830. MBI 11B7CN A. calcoaceticus AC003 32 E. cloacae ECL009 32 E. coliECO002 8 E. faecium EFM001 4 E. faecalis EFS004 4 H. influenzaeHIN002 >128 K. pneumoniae KP001 32 P. aeruginosa PA004 128 P. mirabilisPM002 >128 S. aureus SA009 2 S. marcescens SBSM1 >128 S. pneumoniaeSBSPN3 >128 S. epidermidis SE003 2 S. maltophilia SMA004 128 S. pyogenesSPY006 16 31. MBI 11B7CNR A. calcoaceticus AC002 4 E. cloacaeECL007 64E. coli ECO005 8 E. faecalis EFS001 4 K. pneumoniae KP001 8 P.aeruginosa PA004 64 S. aureus SA093 2 S. epidermidis SE010 4 S.maltophilia SMA002 32 S. marcescens SMS003 >128 32. MBI 11B8CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 16 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA005 >128 S. aureus SA009 4 S. epidermidis SF002 4 S.maltophilia SMA002 128 S. marcescens SMS003 >128 33. MBI 11B9CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 8 E. faeciumEFM002 4 E. faecalis EFS002 8 H. influenzae HIN002 >128 K. pneumoniaeKP001 32 P. aeruginosa PA004 128 P. mirabilis PM002 >128 S. aureus SA0104 S. pneumoniae SBSPN2 >128 S. epidermidis SE010 2 S. maltophilia SMA00232 S. marcescens SMS003 >128 S. pneumoniae SPN044 >128 S. pyogenesSPY005 16 34. MBT 11B9ACN A. calcoaceticus AC001 32 E. cloacaeECL007 >128 E. coli ECO003 8 E. faecium EFM001 4 E. faecalis EFS004 8 K.pneumoniae KP002 32 P. aeruginosa PA005 >128 S. aureus SA019 2 S.epidermidis SE002 2 S. maltophilia SMA001 16 S. marcescens SMS004 >12835. MBI 11B10CN E. faecium EFM003 4 E. faecalis EFS002 64 S. aureusSA008 2 36. MBI 11B16CN A. calcoaceticus AC002 4 E. cloacae ECL007 >128E. coli ECO005 16 E. faecalis EFS001 2 K. pneumoniae KP001 16 P.aeruginosa PA004 >128 S. aureus SA093 2 S. epidemidis SE010 4 S.maltophilia SMA002 32 S. marscescens SMS003 >128 37. MBI 11B17CN A.calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coli ECO005 8 E.faecalis EFS008 4 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S.aureus SA093 2 S. epidermidis SE010 4 S. maltophilial SMA002 32 S.marcescens SMS003 >128 38. MBI 11B18CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS008 4 K. pneunoniaeKP001 32 P. aeruginosa PA004 >128 S. aureus SA093 2 S. epidermidis SE0104 S. maltophilia SMA002 64 S. marcescens SMS003 >128 39. MBT 11C3CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM002 1 E. faecalis EFS002 32 K. pneumoniae KP001 128 P.aeruginosa PA005 >128 S. aureus SA005 2 S. epidermidis SE002 2 S.maltophilia SMA002 64 S. marcescens SMS004 >128 40. MBT 11C4CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 32 E.faecium EFM003 2 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA005 >128 S. aureus SA009 4 S. epidermidis SE002 4 S.maltophilia SMA002 64 S. marcescens SMS004 >128 41. MBI 11C5CN A.calcoaceticus AC001 32 E. cloacae ECL007 >128 E. coli ECO001 8 E.faecium EFM003 2 E. faecalis EFS002 16 K. pneumoniae KP002 16 P.aeruginosa PA003 64 S. aureus SA009 2 S. epidermidis SE002 2 S.maltophilia SMA002 16 S. marcescens SMS004 >128 42. MBI 11D1CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 16 E. faecalis EFS002 32 K. pneumoniae KP002 64 P.aeruginosa PA003 >128 S. aureus SA004 2 S. epidermidis SE010 8 S.maltophilia SMA001 64 S. marcescens SMS003 >128 43. MBI 11D3CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 64 E.faecium EFM003 8 E. faecalis EFS002 32 K. pneumoniae KP002 >128 P.aeruginosa PA024 >128 S. aureus SA009 8 S. maltophilia SMA001 64 S.marcescens SMS004 >128 44. MBI 11D4CN A. calcoaceticus AC001 >64 E.cloacae ECL007 >64 E. coli ECO003 64 E. faecium EFM002 1 E. faecalisEFS002 16 K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S. aureusSA009 4 S. maltophilia SMA001 >64 S. marcescens SMS004 >64 45. MBI11D5CN A. calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coli ECO00364 E. faecium EFM003 1 E. faecalis EFS002 16 K. pneunoniae KP001 >64 P.aeruginosa PA003 >64 S. aureus SA005 8 S. maltophilia SMA001 64 S.marcescens SMS004 >64 46. MBI 11D6CN A. calcoaceticus AC002 4 E. cloacaeECL007 >32 E. coli ECO002 32 E. faecium EFM003 1 E. faecalis EFS002 4 K.pneumoniae KP002 >64 P. aeruginosa PA024 >64 S. aureus SA009 8 S.epidermidis SE010 4 S. maltophpilia SMA001 >64 S. marcescens SMS004 >6447. MBI 11D9M8 E. faecium EFM002 32 S. aureus SA007 32 E. faecalisEFS002 128 S. aureus SA016 128 48. MBI 11D10M8 E. faecium EFM003 32 E.faecalis EFS002 32 S. aureus SA008 32 49. MBI 11D11H A. calcoaceticusAC001 >64 E. cloacae ECL007 >64 E. coli ECO002 32 K. pneumoniaeKP001 >64 P. aeruginosa PA001 >64 S. aureus SA008 4 S. maltophiliaSMA002 >64 S. marcescens SMS004 >64 50. MBT 11D12H A. calcoaceticusAC001 >64 E. cloacae ECL007 >64 E. coli ECO003 64 E. faecalis EFS004 16K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S. aureus SA014 16 S.maltophilia SMA002 >64 S. marcescens SMS004 >64 51. MBI 11D13H A.calcoaceticus AC001 64 E. cloacae ECL007 >64 E. coli ECO002 32 E.faecalis EFS004 16 K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S.aureus SA025 4 S. maltophilia SMA002 >64 S. marcescens SMS004 >64 52.MBI 11DI4CN E. faecium EFM003 1 E. faecalis EFS002 32 S. aureus SA009 453. MBI 11D15CN E. faecium EFM003 4 E. faecalis EFS002 32 S. aureusSA009 8 54. MBI 11D18CN A. calcoaceticus AC003 32 E. cloacae ECL009 64E. coli ECO002 4 E. facium EFM003 2 E. faecalis EFS002 32 H. influenzaeHIN002 >128 K. pneumoniae KP002 64 P. aeruginosa PA006 >128 P. mirabilisPM003 >128 S. aureus SA010 4 P. vulgaris SBPV1 32 S. marcescensSBSM2 >128 S. pneumoniae SBSPN3 64 S. epidermidis SE010 2 S. maltophiliaSMA003 16 S. pyogenes SPY003 32 55. MBI 11E1CN A. calcoaceticus AC001 32E. cloacae ECL007 >128 E. coli ECO003 8 E. faecium EFM001 8 E. faecalisEFS002 8 K. pneumoniae KP002 32 P. aeruginosa PA003 128 S. aureus SA0061 S. maltophilia SMA001 64 S. marcescens SMS003 >128 56. MBI 11E2CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 8 E. faeciumEFM001 16 E. faecalis EFS002 32 K. pneumoniae KP002 64 P. aeruginosaPA001 >128 S. aureus SA016 2 S. epidermidis SE010 4 S. maltophiliaSMA001 64 S. marcescens SMS004 >128 57. MBI 11E3CN A. calcoaceticusAC001 16 E. cloacae ECL007 >128 E. coli ECO001 4 E. faecium EFM003 2 E.faecalis EFS004 8 H. influenzae HIN002 >128 K. pneumoniae KP002 32 P.aeruginosa PA041 64 P. mirabilis PM001 >128 S. aureus SA010 2 S.pneumoniae SBSPN2 >128 S. epidermidis SE002 1 S. maltophilia SMA001 32S. marcescens SMS004 >128 S. pneumoniae SPN044 >128 S. pyogenes SPY00216 58. MBI 11F1CN E. cloacae ECL007 >128 E. coli ECO003 8 E. faeciumEFM003 2 E. faecalis EFS004 16 K. pneumoniae KP002 32 P. aeruginosaPA004 64 S. aureus SA009 2 S. marcescens SBSM1 >128 S. marcescensSMS003 >128 59. MBI 11F2CN A. calcoaceticus AC002 4 E. coli ECO002 8 E.faecium EFM002 4 E. faecalis EFS002 32 K. pneumoniae KP002 128 P.aeruginosa PA005 >128 S. aureus SA012 4 S. epidermidis SE002 4 S.maltophilia SMA002 64 S. marcescens SMS004 >128 60. MBI 11E3CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 8 E. faeciumEFM003 4 E. faecalis EFS002 8 H. influenzae HIN002 >128 K. pneumoniaeKP002 64 P. aeruginosa PA041 128 S. aureus SA005 2 S. pneumoniaeSBSPN3 >128 S. epidermidis SE003 2 S. maltophilia SMA002 64 S.marcescens SMS004 >128 S. pneumoniae SPN044 >128 S. pyogenes SPY006 861. MBI 11F4CN A. calcoaceticus AC003 16 E cloacae ECL006 16 E. coliECO001 8 E. faecalis EFS004 8 H. influenzae HIN003 >128 K. pneumoniaeKP001 8 P. aeruginosa PA020 32 S. aureus SA007 I S. marcescensSBSM1 >128 S. pneumoniae SBSPN3 >128 S. epidermidis SE010 2 S.maltophilia SMA006 16 S. pyogenes SPY005 32 62. MBI 11F4CNR A.calcoaceticus AC002 16 E. cloacae ECL007 32 E. coli ECO005 32 E.faecalis EFS008 32 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S.aureus SA093 8 S. epidermidis SE010 8 S. maltophilia SMA002 32 S.marcescens SMS003 >128 63. MBI 11G2CN E. cloacae ECL007 >128 E. coliECO003 16 E. faecium EFM002 4 E. faecalis EFS004 16 K. pneumoniae KP002128 P. aeruginosa PA004 >128 S. aureus SA009 2 S. maltophiliaSMA001 >128 S. marcescens SMS004 >128 64. MBI 11G3CN E. cloacaeECL007 >128 E. coli ECO003 64 E. faecium EFM002 32 E. faecalis EFS002 64K. pneumoniae KP001 >128 P. aeruginosa PA003 >128 S. aureus SA009 8 S.maltophilia SMA001 >128 S. marcescens SMS004 >128 65. MBI 11G4CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 32 E.faecium EFM003 1 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA004 1 S. epidermidis SE010 2 S.maltophilia SMA002 64 S. marcescens SMS003 >128 66. MBI 11G5CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO003 16 E.faecium EFM002 8 E. faecalis EFS002 16 K. pnuemoniae KP001 >128 P.aeruginosa PA003 >128 S. aureus SA012 4 S. epidermidis SE002 2 S.maltophilia SMA002 64 S. marcescens SMS004 >128 67. MBI 11G6CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 32 E.faecium EFM003 4 E. faecalis EFS002 128 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA006 2 S. epidermidis SE002 8 S.maltophilia SMA001 >128 S. marcescens SMS003 >128 68. MBI 11G6ACN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 64 E.faecalis EFS008 >128 K. pneumoniae KP001 >128 P. aeruginosa PA004 >128S. aureus SA014 64 S epidermidis SE010 32 S. maltophilia SMA002 >128 S.marcescens SMS003 >128 69. MBI 11G7CN A. calcoaceticus AC001 128 E.cloacae ECL006 64 E. coli ECO005 8 E. faecium EFM001 8 E. faecalisEFS002 32 H. influenzae HIN002 >128 K. puenmoniac KP001 16 P. aeruginosaPA006 >128 S. aureus SA012 2 H. influenzae SBHIN2 >128 S. marcescensSBSM1 >128 S. pneumoniae SBSPN2 >128 S. epidermidis SE002 2 S.maltophilia SMA001 32 S. marcescens SMS003 >128 S. pneumoniaeSPN044 >128 S. pyogenes SPY006 16 70. MBI 11G7ACN A. calcoaceticus AC0024 E. cloacae ECL007 >32 E. coli ECO002 16 E. faecium EFM001 8 E.faecalis EFS008 32 K. pneumoniae KP002 >32 P. aeruginosa PA006 >32 S.aureus SA010 1 S. epidermidis SE002 4 S. maltophilia SMA001 32 S.marcescens SMS004 >32 71. MBI 11G13CN E. coli ECO002 32 E. faeciumEFM002 16 E. faecalis EFS002 64 H. influenzae HIN002 >128 P. aeruginosaPA004 >128 S. aureus SA004 4 E. coli SBECO3 32 S. marcescens SBSM1 >128S. pneumoniae SBSPN3 128 72. MBI 11G14CN A. calcoaceticus AC002 8 E.cloacae ECL007 >128 E. coli ECO003 32 E. faecium EFM001 16 E. faecalisEFS002 32 K. pneumoniae KP002 128 P. aeruginosa PA006 >128 S. aureusSA013 0.5 S. epidermidis SE002 8 S. maltophilia SMA002 128 S. marcescensSMS004 >128 73. MBI 11G16CN A. calcoaceticus AC002 8 E. cloacaeECL007 >128 E. coli ECO005 16 E. faecalis EFS008 16 K. pneumoniae KP00116 P. aeruginosa PA004 128 S. aureus SA093 2 S. epidermidis SE010 4 S.maltophilia SMA002 64 S. marcescens SMS003 >128

[0169] Broth Dilution Assay

[0170] This assay also uses calcium and magnesium supplemented MuellerHinton broth as the growth medium. Typically 100 μl of broth isdispensed into each well of a 96-well microtitre plate and 100 μlvolumes of two-fold serial dilutions of the peptide analogue are madeacross the plate. One row of wells receives no peptide and is used as agrowth control. Each well is inoculated with approximately 5×10⁵ CFU ofbacteria and the plate is incubated at 35-37° C. for 16-20 hours. TheMIC is again recorded at the lowest concentration of peptide thatcompletely inhibits growth of the organism as determined by visualinspection.

[0171] For example, MIC values were established for a series of peptideanalogues against S. aureus strains. Results are shown in Table 6 below.TABLE 6 MIC (μg/ml) MBI MBI MBI MBI MBI MBI MBI Organism Organism # 10CN11CN 11A1CN 11A2CN 11B1CN 11B2CN 11B7CN Gram-negative: A. calcoaceticusAC001 64 256 >256 >256 64 128 64 E. cloacae ECL007256 >256 >256 >256 >256 >256 >256 E. coli ECO005 64 128 >256 >256 64 6464 K. pneumoniae KP001 64 >256 >256 >256 >256 >256 256 P. aeruginosaPA004 >256 256 >256 >256 64 256 256 S. maltophilia SMA002 6464 >256 >256 32 32 32 S. marcescensSMS003 >256 >256 >256 >256 >256 >256 >256 Gram-positive: E. faecalisEFS004 64 128 >256 >256 64 64 64 S. aureus SA002 16 64 >256 >256 32 3216 S. epidermidis SE005 8 8 16 256 4 4 4

[0172] Time Kill Assay

[0173] Time kill curves are used to determine the antimicrobial activityof cationic peptides over a time interval. Briefly, in this assay, asuspension of microorganisms equivalent to a 0.5 McFarland Standard isprepared in 0.9% saline. This suspension is then diluted such that whenadded to a total volume of 9 ml of cation-adjusted Mueller Hinton broth,the inoculum size is 1×10⁶ CFU/ml. An aliquot of 0.1 ml is removed fromeach tube at pre-determined intervals up to 24 hours, diluted in 0.9%saline and plated in triplicate to determine viable colony counts. Thenumber of bacteria remaining in each sample is plotted over time todetermine the rate of cationic peptide killing. Generally a three ormore log₁₀ reduction in bacterial counts in the antimicrobial suspensioncompared to the growth controls indicate an adequate bactericidalresponse.

[0174] As shown in FIG. 3, all peptides demonstrated a three or morelog₁₀ reduction in bacterial counts in the antimicrobial suspensioncompared to the growth controls indicating that these peptides have metthe criteria for a bactericidal response.

[0175] Synergy Assay

[0176] Treatment with a combination of peptide analogues andconventional antibiotics can have a synergistic effect. Synergy isassayed using the agarose dilution technique, where an array of plates,each containing a combination of peptide and antibiotic in a uniqueconcentration mix, is inoculated with the bacterial isolates. Synergy isinvestigated for peptide analogues in combination with a number ofconventional antibiotics including, but not limited to, penicillins,cephalosporins, carbapenems, monobactams, aminoglycosides, macrolides,fluoroquinolones.

[0177] Synergy is expressed as a Fractional Inhibitory Concentration(FIC), which is calculated according to the equation below. An FIC ofless than or equal to 0.5 is evidence of synergy, although combinationswith higher values may be therapeutically useful.${FIC} = {\frac{{MIC}\left( {{peptide}\quad {in}\quad {combination}} \right)}{{MIC}\left( {{peptide}\quad {alone}} \right)} + \frac{{MIC}\left( {{antibiotic}\quad {in}\quad {combination}} \right)}{{MIC}\left( {{antibiotic}\quad {alone}} \right)}}$

[0178] Table 7 shows exemplary synergy data for combinations ofindolicidin analogues and Mupirocin. TABLE 7 Mupirocin Mupirocin PeptidePeptide MIC Comb. MIC MIC Comb. MIC Peptide Organism (μg/ml) (μg/ml)(μg/ml) (μg/ml) FIC MBI 11A1CN E. coli ECO1 >100 10 32 4 0.14 MBI 11A1CNE. faecalis EFS8 100 100 >128 >128 2 MBI 11A1CN P. aeruginosaPA3 >100 >100 >128 >128 2 MBI 11A1CN S. aureus SBSA3 100 100 >128 >128 2MBI 11A1CN S. aureus SBSA5 30 10 128 32 0.58 MBI 11A1CN S. marcescensSBSM1 >100 >100 >128 >128 2 MBI 11A3CN E. coli SBECO1 100 30 64 8 0.43MBI 11A3CN E. faecalis EFS8 100 100 >128 >128 2 MBI 11A3CN P. aeruginosaPA3 >100 >100 >128 >128 2 MBI 11A3CN S. aureus SBSA2 >100 >100 128 128 2MBI 11A3CN S. marcescens SBSM2 >100 >100 >128 >128 2 MBI 11B4CN E. coliECO1 >100 10 16 4 0.26 MBI 11B4CN E. faecalis EFS8 100 100 64 64 2 MBI11B4CN S. aureus SBSA3 100 10 32 16 0.60 MBI 11B4CN S. aureusSBSA4 >100 >100 8 8 2 MBI 11B4CN S. marcescens SBSM1 >100 >100 >128 >1282 MBI 11D18CN E. coli SBEC02 >100 10 16 1 0.07 MBI 11D18CN E. faecalisEFS8 100 100 16 16 2 MBI 11D18CN P. aeruginosa PA2 >100 30 128 64 0.53MBI 11D18CN P. aeruginosa PA24 >100 >100 >128 >128 2 MBI 11D18CN P.vulgaris SBPV1 3 3 32 4 1.13 MBI 11D18CN S. aureus SBSA4 >100 0.1 16 20.13 MBI 11D18CN S. marcescens SBSM1 >100 30 >128 64 0.28 MBI 11G13CN E.coli ECO5 100 30 64 8 0.43 MBI 11G13CN P. vulgaris SBPV1 3 3 >128 >128 2MBI 11G13CN P. vulgaris SBPV1 3 3 >128 64 1.25 MBI 11G13CN S. aureusSBSA3 100 100 64 64 2 MBI 11G13CN S. marcescensSBSM1 >100 >100 >128 >128 2

[0179] The MIC values of Mupirocin against strains of E. coli, S.aureus, P. aeruginosa are reduced by at least three fold in combinationwith indolicidin analogues at concentrations that are ≦1/2 MIC value ofthe peptide alone.

[0180] Table 9 shows exemplary synergy data for combinations ofindolicidin analogues and Ciprofloxacin. TABLE 9 Ciprofloxacin PeptideCiprofloxacin Comb. Peptide Comb MIC MIC MIC MIC Peptide Organism(μg/ml) (μg/ml) (μg/ml) (μg/ml) FIC MBI 11D18CN S. aureus SA14 16 8 8 41.00 MBI 11D18CN P. aeruginosa PA24 16 4 >128 16 0.31 MBI 11D18CN S.aureus SA10 32 32 2 2 2.00

[0181] The MIC values of Ciprofloxacin against strains of S. aureus andP. aeruginosa are reduced by at least two fold in combination withindolicidin analogues at concentrations that are ≦1/2 MIC value of thepeptide alone.

Example 5 Biochemical Characterization of Peptide Analogues

[0182] Solubility in Formulation Buffer

[0183] The primary factor affecting solubility of a peptide is its aminoacid sequence. Polycationic peptides are preferably freely soluble inaqueous solutions, especially under low pH conditions. However, incertain formulations, polycationic peptides may form an aggregate thatis removed in a filtration step. As peptide solutions for in vivo assaysare filtered prior to administration, the accuracy and reproducibilityof dosing levels following filtration are examined.

[0184] Peptides dissolved in formulations are filtered through ahydrophilic 0.2 μm filter membrane and then analyzed for total peptidecontent using reversed-phase HPLC. A 100% soluble standard for eachconcentration is prepared by dissolving the peptide in MilliQ water.Total peak area for each condition is measured and compared with thepeak area of the standard in order to provide a relative recovery valuefor each concentration/formulation combination.

[0185] MBI 11CN was prepared in four different buffer systems (A, B, C,and C1) (Table 10, below) at 50, 100, 200 and 400 μg/ml peptideconcentrations. With formulations A or B, both commonly used forsalvation of peptides and proteins, peptide was lost through filtrationin a concentration dependent manner (FIG. 4). Recovery only reached amaximum of 70% at a concentration of 400 μg/ml. In contrast, peptidesdissolved in formulations C and C1 were fully recovered. Bufferscontaining polyanionic ions appear to encourage aggregation, and it islikely that the aggregate takes the form of a matrix which is trapped bythe filter. Monoanionic counterions are more suitable for themaintenance of peptides in a non-aggregated, soluble form, while theaddition of other solubilizing agents may further improve theformulation. TABLE 10 Code Formulation Buffer A PBS 200 mM, pH 7.1 BSodium Citrate 100 mM, pH 5.2 C Sodium Acetate 200 mM, pH 4.6 C1 SodiumAcetate 200 mM/0.5% Polysorbate 80, pH 4.6 D Sodium Acetate 100 mM/0.5%Activated Polysorbate 80, pH 7.5: Lyophilized/Reconstituted

[0186] Solubility in Broth

[0187] The solubility of peptide analogues is assessed in calcium andmagnesium supplemented Mueller Hinton broth by visual inspection. Theprocedure employed is that used for the broth dilution assay except thatbacteria are not added to the wells. The appearance of the solution ineach well is evaluated according to the scale: (a) clear, noprecipitate, (b) light diffuse precipitate and (c) cloudy, heavyprecipitate. Results show that, for example, MBI 10CN is less solublethan MBI 11CN under these conditions and that MBI 11BCN analogues areless soluble than MBI 11ACN analogues.

[0188] Reversed Phase HPLC Analysis of Peptide Analogue Formulations

[0189] Reversed-phase HPLC, which provides an analytical method forpeptide quantification, is used to examine peptides in two differentformulations. A 400 μg/mL solution of MBI 11CN prepared in formulationsC1 and D is analyzed by using a stepwise gradient to resolve freepeptide from other species. Standard chromatographic conditions are usedas follows:

[0190] Solvent A: 0.1% trifluoroacetic acid (TFA) in water

[0191] Solvent B: 0.1% TFA/95% acetonitrile in water

[0192] Media: POROSO® R2-20 (polystyrene divinylbenzene)

[0193] As shown in FIG. 5, MBI 11CN could be separated in two forms, asfree peptide in formulation C1, and as a principally formulation-complexpeptide in formulation D. This complex survives the separation protocolin gradients containing acetonitrile, which might be expected to disruptthe stability of the complex. A peak corresponding to a small amount(<10%) of free peptide is also observed in formulation D. If the shapeof the elution gradient is changed, the associated peptide elutes as abroad low peak, indicating that complexes of peptide in the formulationare heterogeneous.

Example 6 Structural Analysis of Indolicdin Variants Using CircularDichroism Spectroscopy

[0194] Circular dichroism (CD) is a spectroscopic technique thatmeasures secondary structures of peptides and proteins in solution, seefor example, R. W. Woody, (Methods in Enzymology, 246: 34, 1995). The CDspectra of α-helical peptides is most readily interpretable due to thecharacteristic double minima at 208 and 222 nm. For peptides with othersecondary structures however, interpretation of CD spectra is morecomplicated and less reliable. The CD data for peptides is used torelate solution structure to in vitro activity.

[0195] CD measurements of indolicidin analogues are performed in threedifferent aqueous environments, (1) 10 mM sodium phosphate buffer, pH7.2, (2) phosphate buffer and 40% (v/v) trifluoroethanol (TFE) and (3)phosphate buffer and large (100 nm diameter) unilamellar phospholipidvesicles (liposomes) (Table 11). The organic solvent TFE and theliposomes provide a hydrophobic environment intended to mimic thebacterial membrane where the peptides are presumed to adopt an activeconformation.

[0196] The results indicate that the peptides are primarily unordered inphosphate buffer (a negative minima at around 200 nm) with the exceptionof MBI 11F4CN, which displays an additional minima at 220 nm (seebelow). The presence of TFE induces β-turn structure in MBI 11 and MBI11G4CN, and increases α-helicity in MBI 11F4CN, although most of thepeptides remain unordered. In the presence of liposomes, peptides MBI11CN and MBI 11B7CN, which are unordered in TFE, display β-turnstructure (a negative minima at around 230 nm) (FIG. 6). Hence,liposomes appear to induce more ordered secondary structure than TFE.

[0197] A β-turn is the predominant secondary structure that appears in ahydrophobic environment, suggesting that it is the primary conformationin the active, membrane-associated form. In contrast, MBI 11F4CNdisplays increased α-helical conformation in the presence of TFE.Peptide MBI 11F4CN is also the most insoluble and hemolytic of thepeptides tested, suggesting that α-helical secondary structure mayintroduce unwanted properties in these analogues.

[0198] Additionally CD spectra are recorded for APS-modified peptides(Table 11). The results show that these compounds have significantβ-turn secondary structure in phosphate buffer, which is only slightlyaltered in TFE.

[0199] Again, the CD results suggest that a β-turn structure (i.e.membrane-associated) is the preferred active conformation among theindolicidin analogues tested. TABLE 11 Phosphate buffer Conformation TFEConformation Peptide min λ max λ in buffer min λ max λ in TFE MBI 10CN201 — Unordered 203 ˜219  Unordered MBI 11 199 — Unordered 202, 227 220β-turn MBI 11ACN 199 — Unordered 203 219 Unordered MBI 11CN 200 —Unordered 200 — Unordered MBI 11CNY1 200 — Unordered 200 — Unordered MBI11B1CNW1 201 — Unordered 201 — Unordered MBI 11B4ACN 200 — Unordered 200— Unordered MBI 11B7CN 200 — Unordered 204, ˜219 Unordered MBI 11B9ACN200 — Unordered 200 — Unordered MBI 11B9CN 200 — Unordered 200 —Unordered MBI 11D1CN 200 — Unordered 204 — Unordered MBI 11E1CN 201 —Unordered 201 — Unordered MBI 11E2CN 200 — Unordered 201 — Unordered MBI11E3CN 202 226 ppII helix 200 — Unordered MBI 11F3CN 199 228 ppII helix202 — Unordered MBI 11F4CN 202, 220 — Unordered 206, 222 — slightα-helix MBI 11G4CN 199, 221 — Unordered 201, 226 215 β-turn MBI 11G6ACN200 — Unordered 199 — Unordered MBI 11G7ACN 200 — Unordered 202 221Unordered

[0200] TABLE 12 APS-modified Phosphate buffer Conformation TFEConformation peptide min λ max λ in buffer min λ max λ in TFE MBI 11CN202, 229 220 β-turn 203 223 β-turn MBI 11BCN 200, 229 — β-turn 202 222β-turn MBI 11B7CN 202, 230 223 β-turn 199 230 β-turn MBI 11E3CN 202, 229220 β-turn 199 — β-turn MBI 11F3CN 205 — ppII helix 203 230 ppII helix

Example 7 Membrane Permeabilization Assays

[0201] Liposome Dye Release

[0202] A method for measuring the ability of peptides to permeabilizephospholipid bilayers is described (Parente et al., Biochemistry, 29,8720, 1990) Briefly, liposomes of a defined phospholipid composition areprepared in the presence of a fluorescent dye molecule. In this example,a dye pair consisting of the fluorescent molecule8-aminonapthalene-1,3,6-trisulfonic acid (ANTS) and its quenchermolecule p-xylene-bis-pyridinium bromide (DPX) are used. The mixture offree dye molecules, dye free liposomes, and liposomes containingencapsulated ANTS-DPX are separated by size exclusion chromatography. Inthe assay, the test peptide is incubated with the ANTS-DPX containingliposomes and the fluorescence due to ANTS release to the outside of theliposome is measured over time.

[0203] Using this assay, peptide activity, measured by dye release, isshown to be extremely sensitive to the composition of the liposomes atmany liposome to peptide ratios (L/P) (FIG. 7). Specifically, additionof cholesterol to liposomes composed of egg phosphotidylcholine (PC)virtually abolishes membrane permeabilizing activity of MBI 11CN, evenat very high lipid to peptide molar ratios (compare with egg PCliposomes containing no cholesterol). This in vitro selectivity maymimic that observed in vitro for bacterial cells in the presence ofmammalian cells.

[0204] In addition, there is a size limitation to the membranedisruption induced by MBI 11CN. ANTS/DPX can be replaced withfluorescein isothiocyanate-labeled dextran (FD-4), molecular weight4,400, in the egg PC liposomes. No increase in FD-4 fluorescence isdetected upon incubation with MBI 11CN. These results indicate that MBI11CN-mediated membrane disruption allows the release of the relativelysmaller ANTS/DPX molecules (˜400 Da), but not the bulkier FD-4molecules.

[0205]E. coli ML-35 Inner Membrane Assay

[0206] An alternative method for measuring peptide-membrane interactionuses the E. coli strain ML-35 (Lehrer et al., J. Clin. Invest., 84: 553,1989), which contains a chromosomal copy of the lacZ gene encodingβ-galactosidase and is permease deficient. This strain is used tomeasure the effect of peptide on the inner membrane through release ofβ-galactosidase into the periplasm. Release of β-galactosidase ismeasured by spectrophotometrically monitoring the hydrolysis of itssubstrate o-nitrophenol β-D-galactopyranoside (ONPG). The maximum rateof hydrolysis (V_(max)) is determined for aliquots of cells taken atvarious growth points.

[0207] A preliminary experiment to determine the concentration ofpeptide required for maximal activity against mid-log cells, diluted to4×10⁷ CFU/ml, yields a value of 50 μg/ml, which is used in allsubsequent experiments. Cells are grown in two different growth media,Terrific broth (TB) and Luria broth (LB) and equivalent amounts of cellsare assayed during their growth cycles. The resulting activity profileof MBI 11B7CN is shown in FIG. 8. For cells grown in the enriched TBmedia, maximum activity occurs at early mid-log (140 min), whereas forcells grown in LB media, the maximum occurs at late mid-log (230 min).Additionally, only in LB, a dip in activity is observed at 140 min. Thisdrop in activity may be related to a transition in metabolism, such as arequirement for utilization of a new energy source due to depletion ofthe original source, which does not occur in the more enriched TB media.A consequence of a metabolism switch would be changes in the membranepotential.

[0208] To test whether membrane potential has an effect on peptideactivity, the effect of disrupting the electrochemical gradient usingthe potassium ionophore valinomycin is examined. Cells pre-incubatedwith valinomycin are treated with peptide and for MBI 10CN and MBI 11CNONPG hydrolysis diminished by approximately 50% compared to nopre-incubation with valinomycin (FIG. 9). Another cationic peptide thatis not sensitive to valinomycin is used as a positive control.

[0209] Further delineation of the factors influencing membranepermeabilizing activity are tested. In an exemplary test, MBI 11B7CN ispre-incubated with isotonic HEPES/sucrose buffer containing either 150mM sodium chloride (NaCl) or 5 mM magnesium ions (Mg²⁺) and assayed asdescribed earlier. In FIG. 10, a significant inhibition is observed witheither solution, suggesting involvement of electrostatic interactions inthe permeabilizing action of peptides.

Example 8 Erythtocyte Lysis by Indolicidin Analogues

[0210] A red blood cell (RBC) lysis assay is used to group peptidesaccording to their ability to lyse RBC under standardized conditionscompared with MBI 11CN and Gramicidin-S. Peptide samples and washedsheep RBC are prepared in isotonic saline with the final pH adjusted tobetween 6 and 7. Peptide samples and RBC suspension are mixed togetherto yield solutions that are 1% (v/v) RBC and 5, 50 or 500 μg/ml peptide.Assay mixtures are incubated for 1 hour at 37° C. with constant shaking,centrifuged, and the supernatant is measured for absorbance at 540 nm,which detects released hemoglobin. The percentage of released hemoglobinis determined by comparison with a set of known standards lysed inwater. Each set of assays also includes MBI 11CN (500 μg/ml) andGramicidin-S (5 μg/ml) as “low lysis” and “high lysis” controls,respectively.

[0211] MBI-11B7CN-HCl, MBI-11F3CN-HCl and MBI-11F4CN-HCl are testedusing this procedure and the results are presented in Table 13 below.TABLE 13 % lysis at % lysis at % lysis at Peptide 5 μg/ml 50 μg/ml 500μg/ml MBI 11B7CN-HCl 4 13 46 MBI 11F3CN-HCl 1 6 17 MBI 11F4CN-HCl 4 3238 MBI 11CN-TFA N/D N/D 9 Gramicidin-S 30 N/D N/D

[0212] Peptides that at 5 μg/ml lyse RBC to an equal or greater extentthan Gramicidin-S, the “high lysis” control, are considered to be highlylytic. Peptides that at 500 μg/ml lyse RBC to an equal to or lesserextent than MBI 11CN, the “low lysis” control, are considered to benon-lytic. The three analogues tested are all “moderately lytic” as theycause more lysis than MBI 11CN and less than Gramicidin-S. In additionone of the analogues, MBI-11F3CN-HCl, is significantly less lytic thanthe other two variants at all three concentrations tested.

Example 9 Production of Antibodies to Peptide Analogues

[0213] Multiple antigenic peptides (MAPs), which contain four or eightcopies of the target peptide linked to a small non-immunogenic peptidylcore, are prepared as immunogens. Alternatively, the target peptide isconjugated to bovine serum albumin (BSA) or ovalbumin. For example, MBI11CN and its seven amino acid N-terminal and C-terminal fragments areused as target peptide sequences. The immunogens are injectedsubcutaneously into rabbits using standard protocols (see, Harlow andLane, Antibodies: A Laboratory Mantial, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988). After repeated boosters (usuallymonthly), serum from a blood sample is tested in an ELISA against thetarget peptide. A positive result indicates the presence of antibodiesand further tests determine the specificity of the antibody binding tothe target peptide. Purified antibodies can then be isolated from thisserum and used in ELISAs to selectively identify and measure the amountof the target peptide in research and clinical samples.

Example 10 Pharmacology of Peptide Analogues in Plasma and Blood

[0214] The in vitro lifetime of free peptide analogues in plasma and inblood is determined by measuring the amount of peptide present after setincubation times. Blood is collected from sheep, treated with ananticoagulant (not heparin) and, for plasma preparation, centrifuged toremove cells. Formulated peptide is added to either the plasma fractionor to whole blood and incubated. Following incubation, peptide isidentified and quantified directly by reversed phase HPLC. Extraction isnot required as the free peptide peak does not overlie any peaks fromblood or plasma.

[0215] A 1 mg/mL solution of MBI 11CN in formulations C1 and D is addedto freshly prepared sheep plasma at a final peptide concentration of 100μg/mL and incubated at 37° C. At various times, aliquots of plasma areremoved and analyzed for free peptide by reversed phase HPLC. From eachchromatogram, the area of the peak corresponding to free peptide isintegrated and plotted against time of incubation. As shown in FIG. 11,peptide levels diminish over time. Moreover, when administered informulation D, up to 50% of the peptide is immediately released fromformulation-peptide complex on addition to the blood. The decay curvefor free peptide yields an apparent half-life in blood of 90 minutes forboth formulation C1 and D. These results indicate that in sheep's bloodMBI 11CN is relatively resistant to plasma peptidases and proteases. Newpeaks that appeared during incubation may be breakdown products of thepeptide.

[0216] Peptide levels in plasma in vivo are measured after iv or ipadministration of 80-100% of the maximum tolerated dose of peptideanalogue in either formulation C1 or D. MBI 11CN in formulation C1 isinjected intravenously into the tail vein of CD1 ICRBR strain mice. Atvarious times post-injection, mice are anesthetized and blood is drawnby cardiac puncture. Blood from individual mice is centrifuged toseparate plasma from cells. Plasma is then analyzed by reversed phaseHPLC column. The resulting elution profiles are analyzed for freepeptide content by UV absorbance at 280 nm, and these data are convertedto concentrations in blood based upon a calibrated standard. Each datapoint represents the average blood level from two mice. In this assay,the detection limit is approximately 1 μg/ml, less than 3% of the doseadministered

[0217] The earliest time point at which peptide can be measured is threeminutes following injection, thus, the maximum observed concentration(in μg/ml) is extrapolated back to time zero (FIG. 12). The projectedinitial concentration corresponds well to the expected concentration ofbetween 35 and 45 μg/ml. Decay is rapid, however, and when the curve isfitted to the equation for exponential decay, free circulating peptideis calculated to have a half life of 2.1 minutes. Free circulatingpeptide was not detectable in the blood of mice that were injected withMBI 11CN in formulation D, suggesting that peptide is not released asquickly from the complex as in vitro.

[0218] In addition, MBI 11CN is also administered to CD1 ICRBR strainmice by a single ip injection at an efficacious dose level of 40 mg/kg.Peptide is administered in both formulations C1 and D to determine ifpeptide complexation has any effect on blood levels. At various timespost injection, mice are anesthetized and blood is drawn by cardiacpuncture. Blood is collected and analyzed as for the iv injection.

[0219] MBI 11CN administered by this route demonstrated a quitedifferent pharmacologic profile (FIG. 13). In formulation C1, peptideentered the blood stream quickly, with a peak concentration of nearly 5μg/ml after 15 minutes, which declined to non-detectable levels after 60minutes. In contrast, peptide in formulation D is present at a levelabove 2 μg/ml for approximately two hours. Therefore, formulationaffects entry into, and maintenance of levels of peptide in the blood.

Example 11 Toxicity of Peptide Analogues In vivo

[0220] The acute, single dose toxicity of various indolicidin analoguesis tested in Swiss CD1 mice using various routes of administration. Inorder to determine the inherent toxicities of the peptide analogues inthe absence of any formulation/delivery vehicle effects, the peptidesare all administered in isotonic saline with the final pH between 6 and7.

[0221] Intraperitoneal route. Groups of 6 mice are injected with peptidedoses of between 80 and 5 mg/kg in 500 μl dose volumes. After peptideadministration, the mice are observed for a period of 5 days, at whichtime the dose causing 50% mortality (LD₅₀), the dose causing 90-100%mortality (LD₉₀₋₁₀₀) and maximum tolerated dose (MTD) levels aredetermined. The LD₅₀ values are calculated using the method of Reed andMuench (J. of Amer. Hyg. 27: 493-497, 1938). The results presented inTable 14 show that the LD₅₀ values for MBI 11CN and analogues range from21 to 52 mg/kg. TABLE 14 Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI 11CN 34 mg/kg 40mg/kg 20 mg/kg MBI 11B7CN 52 mg/kg >80 mg/kg   30 mg/kg MBI 11E3CN 21mg/kg 40 mg/kg <20 mg/kg   MBI 11F3CN 52 mg/kg 80 mg/kg 20 mg/kg

[0222] Intravenous route. Groups of 6 mice are injected with peptidedoses of 20, 16, 12, 8, 4 and 0 mg/kg in 100 μl volumes (4 ml/kg). Afteradministration, the mice are observed for a period of 5 days, at whichtime the LD₅₀, LD₉₀₋₁₀₀ and MTD levels are determined. The results fromthe IV toxicity testing of MBI 11CN and three analogues are shown inTable 15. The LD₅₀, LD₉₀₋₁₀₀ and MTD values range from 5.8 to 15 mg/kg,8 to 20 mg/kg and <4 to 12 mg/kg respectively. TABLE 15 Peptide LD₅₀LD₉₀₋₁₀₀ MTD MBI 11CN HCl 5.8 mg/kg 8.0 mg/kg <4 mg/kg MBI 11B7CN HCl7.5 mg/kg 16 mg/kg 4 mg/kg MBI 11F3CN HCl 10 mg/kg 12 mg/kg 8 mg/kg MBI11F4CN HCl 15 mg/kg 20 mg/kg 12 mg/kg

[0223] Subcutaneous route. The toxicity of MBI 11CN is also determinedafter subcutaneous (SC) administration. For SC toxicity testing, groupsof 6 mice are injected with peptide doses of 128, 96, 64, 32 and 0 mg/kgin 300 μL dose volumes (12 mL/kg). After administration, the mice areobserved for a period of 5 days. None of the animals died at any of thedose levels within the 5 day observation period. Therefore, the LD₅₀,LD₉₀₋₁₀₀ and MTD are all taken to be greater than 128 mg/kg. Micereceiving higher dose levels showed symptoms simlar to those seen afterIV injection suggesting that peptide entered the systemic circulation.These symptoms are reversible, disappearing in all mice by the secondday of observations.

[0224] The single dose toxicity of MBI 10CN and MBI 11CN in differentformulations is also examined in outbred ICR mice (Table 16).Intraperitoneal injection (groups of 2 mice) of MBI 10CN in formulationD show no toxicity up to 29 mg/kg and under the same conditions MBI 11CNshow no toxicity up to 40 mg/kg.

[0225] Intravenous injection (groups of 10 mice) of MBI 10CN informulation D show a maximum tolerated dose (MTD) of 5.6 mg/kg (Table16). Injection of 11 mg/kg gave 40% toxicity and 22 mg/kg result in 100%toxicity. Intravenous injection of MBI 11CN in formulation C(lyophilized) show a MTD of 3.0 mg/kg. Injection at 6.1 mg/kg result in10% toxicity and at 12 mg/kg 100% toxicity. TABLE 16 MTD Peptide Route #Animals Formulation (mg/kg) MBI 10CN ip 2 formulation D >29 MBI 11CN ip2 formulation D >40 MBI 10CN iv 10 formulation D 5.6 MBI 11CN iv 10formulation C 3.0 (lyophilized)

[0226] These results are obtained using peptide/buffer solutions thatare lyophilized after preparation and reconstituted with water. If thepeptide solution is not lyophilized before injection, but usedimmediately after preparation, an increase in toxicity is seen, and themaximum tolerated dose can decrease by up to four-fold. For example, anintravenous injection of MBI 11CN as a non-lyophilized solution,formulation C1, at 1.5 mg/kg results in 20% toxicity and at 3.0 mg/kggave 100% toxicity. HPLC analyses of the non-lyophilized and lyophilizedformulations indicate that the MBI 11CN forms a complex withpolysorbate, and this complexation of the peptide reduces its toxicityin mice.

[0227] In addition, mice are multiply injected by an intravenous routewith MBI 11CN (Table 17). In one representative experiment, peptideadministered in 10 injections of 0.84 mg/kg at 5 minute intervals is notlethal. However, two injections of peptide at 4.1 mg/kg administeredwith a 10 minute interval results in 60% mortality. TABLE 17 # DoseInjec- Time Peptide Route Formulation Level* tions Interval Result MBI11CN iv formulation 0.84 10  5 min no D mortality MBI 11CN ivformulation 4.1   2 10 min 66% D mortality

[0228] To assess the impact of dosing mice with peptide analogue, aseries of histopathology investigations can be carried out. Groups ofmice are administered analogue at dose levels that are either at, orbelow the MTD, or above the MTD, a lethal dose. Multiple injections maybe used to mimic possible treatment regimes. Groups of control mice arenot injected or injected with buffer only.

[0229] Following injection, mice are sacrificed at specified times andtheir organs immediately placed in a 10% balanced formalin solution.Mice that die as a result of the toxic effects of the analogue also havetheir organs preserved immediately. Tissue samples are taken andprepared as stained micro-sections on slides which are then examinedmicroscopically. Damage to tissues is assessed and this information canbe used to develop improved analogues, improved methods ofadministration or improved dosing regimes.

Example 12 In vivo Efficacy of Peptide Analogues

[0230] Analogues are tested for their ability to rescue mice from lethalbacterial infections. The animal model used is an intraperitoneal (ip)inoculation of mice with 10⁶-10⁸ Gram-positive organisms with subsequentadministration of peptide. The three pathogens investigated,methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus(MRSA), or S. epidermidis are injected ip into mice. For untreated mice,death occurs within 12-18 hours with MSSA and S. epidermis and within6-10 hours with MRSA.

[0231] Peptide is administered by two routes, intraperitoneally, at onehour post-infection, or intravenously, with single or multiple dosesgiven at various times pre- and post-infection.

[0232] MSSA infection. In a typical protocol, groups of 10 mice areinfected intraperitoneally with a LD₉₀₋₁₀₀ dose (5.2×10⁶ CFU/mouse) ofMSSA (Smith, ATCC #19640) injected in brain-heart infusion containing 5%mucin. This strain of S. aureus is not resistant to any commonantibiotics. At 60 minutes post-infection, MBI 10CN or MBI 11CN, informulation D, is injected intraperitoneally at the stated dose levels.An injection of formulation alone serves as a negative control andadministration of ampicillin serves as a positive control. The survivalof the mice is monitored at 1, 2, 3 and 4 hrs post-infection and twicedaily thereafter for a total of 8 days.

[0233] As shown in FIG. 14, MBI 10CN is maximally active against MSSA(70-80% survival) at doses of 14.5 to 38.0 mg/kg, although 100% survivalis not achieved. Below 14.5 mg/kg, there is clear dose-dependentsurvival. At these lower dose levels, there appears to be ananimal-dependent threshold, as the mice either die by day 2 or survivefor the full eight day period. As seen in FIG. 15, MBI 11CN, on theother hand, rescued 100% of the mice from MSSA infection at a dose levelof 35.7 mg/kg, and was therefore as effective as ampicillin. There waslittle or no activity at any of the lower dose levels, which indicatesthat a minimum bloodstream peptide level must be achieved during thetime that bacteria are a danger to the host.

[0234] As shown above, blood levels of MBI 11CN can be sustained at alevel of greater than 2 μ/ml for a two hour period inferring that thisis higher than the minimum level.

[0235] Additionally, eight variants based on the sequence of MBI 11CNare tested against MSSA using the experimental system described above.Peptides prepared in formulation D are administered at dose levelsranging from 12 to 24 mg/kg and the survival of the infected mice ismonitored for eight days (FIGS. 16-24). The percentage survival at theend of the observation period for each variant is summarized in Table18. As shown in the table, several of the variants showed efficacygreater than or equal to MBI 11CN under these conditions. TABLE 18 %Survival 24 mg/kg 18 mg/kg 12 mg/kg 100 90 11B1CN, 11F3CN 80 70 11E3CN60 11B7CN 50 11CN 40 11G2CN 30 11B1CN 20 11G4CN 10 11CN, 11B7CN, 11G2CN11B8CN, 11F3CN 0 11A1CN 11A1CN, 11G2CN, 11CN, 11A1CN, 11G4CN 11B1CN,11B7CN, 11B8CN, 11F3CN, 11G4CN

[0236]S. epidermidis infection. Peptide analogues generally have lowerMIC values against S. epidermidis in vitro, therefore, lower bloodpeptide levels might be more effective against infection.

[0237] In a typical protocol, groups of 10 mice are injectedintraperitoneally with an LD₉₀₋₁₀₀ dose (2.0×10⁸ CFU/mouse) of S.epidermidis (ATCC #12228) in brain-heart infusion broth containing 5%mucin. This strain of S. epidermidis is 90% lethal after 5 days. At 15mins and 60 mins post-infection, various doses of MBI 11CN informulation D are injected intravenously via the tail vein. An injectionof formulation only serves as the negative control and injection ofgentamicin serves as the positive control; both are injected at 60minutes post-infection. The survival of the mice is monitored at 1, 2,3, 4, 6 and 8 hrs post-infection and twice daily thereafter for a totalof 8 days.

[0238] As shown in FIGS. 25A and 25B, MBI 11CN prolongs the survival ofthe mice. Efficacy is observed at all three dose levels with treatment15 minutes post-infection, however, there is less activity at 30 minutespost-infection and no significant effect at 60 minutes post-infection.Time of administration appears to be important in this model system,with a single injection of 6.1 mg/kg 15 minutes post-infection givingthe best survival rate.

[0239] MRSA infection. MRSA infection, while lethal in a short period oftime, requires a much higher bacterial load than MSSA. In a typicalprotocol, groups of 10 mice are injected intraperitoneally with aLD₉₀₋₁₀₀ dose (4.2×10⁷ CFU/mouse) of MRSA (ATCC #33591) in brain-heartinfusion containing 5% mucin. The treatment protocols are as follows,with the treatment times relative to the time of infection: 0 mg/kgFormulation D alone (negative control), injected at 0 mins 5 mg/kg Three5.5 mg/kg injections at −5, +55, and +115 mins 1 mg/kg (2 hr) Five 1.1mg/kg injections at −5, +55, +115, +175 and +235 mins 1 mg/kg (20 min)Five 1.1 mg/kg injections at −10, −5, 0, +5, and +10 mins Vancomycin(positive control) injected at 0 mins

[0240] MBI 11CN is injected intravenously in the tail vein informulation D. Survival of mice is recorded at 1, 2, 3, 4, 6, 8, 10, 12,20, 24 and 30 hrs post-infection and twice daily thereafter for a totalof 8 days. There was no change in the number of surviving mice after 24hrs (FIG. 26).

[0241] The 1 mg/kg (20 min) treatment protocol, with injections 5minutes apart centered on the infection time, delayed the death of themice to a significant extent with one survivor remaining at the end ofthe study. The results presented in Table 19 suggest that a sufficientlyhigh level of MBI 11CN maintained over a longer time period wouldincrease the number of mice surviving. The 5 mg/kg and 1 mg/kg (2 hr)results, where there is no improvement in survivability over thenegative control, indicates that injections 1 hour apart, even at ahigher level, are not effective against MRSA. TABLE 19 Time ofObservation Percentage of Animals Surviving (Hours post-infection) NoTreatment Treatment 6  50% 70% 8 0 40% 10 0 30% 12 0 20%

Example 13 Activation of Polysorbate 80 by Ultraviolet Light

[0242] A solution of 2% (w/w) polysorbate 80 is prepared in water andplaced in a suitable reaction vessel, such as a quartz cell. Othercontainers that are UV translucent or even opaque can be used ifprovision is made for a clear light path or an extended reaction time.In addition, the vessel should allow the exchange of air but minimizeevaporation.

[0243] The solution is irradiated with ultraviolet light using a lampemitting at 254 nm. Irradiation can also be performed using a lampemitting at 302 nm. The activation is complete in 1-14 days dependingupon the container, the depth of the solution, and air exchange rate.The reaction is monitored by a reversed-phased HPLC assay, whichmeasures the formation of APS-modified MBI 11CN when the light-activatedpolysorbate is reacted with MBI 11CN.

[0244] Some properties of activated polysorbate are determined. Becauseperoxides are a known by-product of exposing ethers to UV light,peroxide formation is examined through the effect of reducing agents onthe activated polysorbate. As seen in FIG. 27A, activated polysorbatereadily reacts with MBI 11CN. Pre-treatment with 2-mercaptoethanol (FIG.27B), a mild reducing agent, eliminates detectable peroxides, but doesnot cause a loss of conjugate forming ability. Treatment with sodiumborohydride (FIG. 27C), eliminates peroxides and eventually eliminatesthe ability of activated polysorbate to modify peptides. Hydrolysis ofthe borohydride in water raises the pH and produces borate as ahydrolysis product. However, neither a pH change nor borate areresponsible.

[0245] These data indicate that peroxides are not involved in themodification of peptides by activated polysorbate. Sodium borohydrideshould not affect epoxides or esters in aqueous media, suggesting thatthe reactive group is an aldehyde or ketone. The presence of aldehydesin the activated polysorbate is confirmed by using a formaldehyde test,which is specific for aldehydes including aldehydes other thanformaldehyde.

[0246] Furthermore, activated polysorbate is treated with2,4-dinitrophenylhydrazine (DNPH) in an attempt to capture the reactivespecies. Three DNPH-tagged components are purified and analyzed by massspectroscopy. These components are polysorbate-derived with molecularweights between 1000 and 1400. This indicates that low molecular weightaldehydes, such as formaldehyde or acetaldehyde, are involved.

Example 14 Formation of APS-Modified Peptides

[0247] APS-modified peptides are prepared either in solid phase orliquid phase. For solid phase preparation, 0.25 ml of 4 mg/ml of MBI11CN is added to 0.5 ml of 0.4 M Acetic acid-NaOH pH 4.6 followed byaddition of 0.25 ml of UV-activated polysorbate. The reaction mix isfrozen by placing it in a −80° C. freezer. After freezing, the reactionmix is lyophilized overnight.

[0248] For preparing the conjugates in an aqueous phase, a sample of UVactivated polysorbate 80 is first adjusted to a pH of 7.5 by theaddition of 0.1M NaOH. This pH adjusted solution (0.5 ml) is added to1.0 ml of 100 mM sodium carbonate, pH 10.0, followed immediately by theaddition of 0.5 ml of 4 mg/ml of MBI 11CN. The reaction mixture isincubated at ambient temperature for 22 hours. The progress of thereaction is monitored by analysis at various time points using RP-HPLC(FIG. 28). In FIG. 28, peak 2 is unreacted peptide, peak 3 isAPS-modified peptide. Type 1 is the left-most of peak 3 and Type 2 isthe right-most of peak 3.

[0249] Table 20 summarizes data from several experiments. Unlessotherwise noted in table 20, the APS-modified peptides are prepared viathe lyophilization method in 200 mM acetic acid-NaOH buffer, pH 4.6.TABLE 20 COMPLEX SEQUENCE NAME TYPE 1 TYPE 2 ILKKWPWWPWRRKamide 11CNSolid phase, pH 2.0 Yes Low Solid phase, pH 4.6 Yes Yes Solid phase, pH5.0 Yes Yes Solid phase, pH 6.0 Yes Yes Solid phase, pH 8.3 Yes YesSolution, pH 2.0 Trace Trace Solution, pH 10.0 Yes Yes-Slow(Ac)₄-ILKKWPWWPWRRKamide 11CN-Y1 No No ILRRWPWWPWRRKamide 11B1CN YesLowered ILRWPWWPWRRKamide 11B7CN Yes Lowered ILWPWWPWRRKamide 11B8CN YesLowered ILRRWPWWPWRRRamide 11B9CN Yes Trace ILKKWPWWPWKKKamide 11B10CNYes Yes iLKKWPWWPWRRkamide 11E3CN Yes Yes ILKKWVWWPWRRKamide 11F3CN YesYes ILKKWPWWPWKamide 11G13CN Yes Yes ILKKWPWWPWRamide 11G14CN Yes Trace

[0250] The modification of amino groups is further analyzed bydetermining the number of primary amino groups lost during attachment.The unmodified and modified peptides are treated with2,4,6-trinitrobenzenesulfonic acid (TNBS) (R. L. Lundblad in Techniquesin Protein Modification and Analysis pp. 151-154, 1995) (Table 21).

[0251] Briefly, a stock solution of MBI 11CN at 4 mg/ml and an equimolarsolution of APS-modified MBI 11CN are prepared. A 0.225 ml aliquot ofMBI 11CN or APS-modified MBI 11CN is mixed with 0.225 ml of 200 mMsodium phosphate buffer, pH 8.8. A 0.450 ml aliquot of 1% TNBS is addedto each sample, and the reaction is incubated at 37° C. for 30 minutes.The absorbance at 367 nm is measured, and the number of modified primaryamino groups per molecule is calculated using an extinction coefficientof 10,500 M⁻¹ cm⁻¹ for the trinitrophenyl (TNP) derivatives.

[0252] The primary amino group content of the parent peptide is thencompared to the corresponding APS-modified peptide. As shown below, theloss of a single primary amino group occurs during formation of modifiedpeptide. Peptides possessing a 3,4 lysine pair consistently give resultsthat are 1 residue lower than expected, which may reflect sterichindrance after titration of one member of the doublet. TABLE 21TNP/APS- modified PEPTIDE SEQUENCE TNP/PEPTIDE peptide CHANGEILKKWPWWPWRRKamide 2.71 1.64 1.07 ILRRWPWWPWRRKamide 1.82 0.72 1.10IlKKWPWWPWRRkamide 2.69 1.61 1.08 ILKKWVWWPWRRKamide 2.62 1.56 1.06

[0253] Stability of APS-Modified Peptide Analogues

[0254] APS-modified peptides demonstrate a high degree of stabilityunder conditions that promote the dissociation of ionic or hydrophobiccomplexes. APS-modified peptide in formulation D is prepared as 800μg/ml solutions in water, 0.9% saline, 8M urea, 8M guanidine-HCl, 67%1-propanol, 1M HCl and 1M NaOH and incubated for 1 hour at roomtemperature. Samples are analyzed for the presence of free peptide usingreversed phase HPLC and the following chromatographic conditions:

[0255] Solvent A: 0.1% trifluoroacetic acid (TFA) in water

[0256] Solvent B: 0.1% TFA/95% acetonitrile in water

[0257] Media: POROS R2-20 (polystyrene divinylbenzene)

[0258] Elution: 0% B for 5 column volumes

[0259] 0-25% B in 3 column volumes

[0260] 25% B for 10 column volumes

[0261] 25-95% B in 3 column volumes

[0262] 95% B for 10 column volumes

[0263] Under these conditions, free peptide elutes exclusively duringthe 25% B step and formulation-peptide complex during the 95% B step.None of the dissociating conditions mentioned above, with the exceptionof 1M NaOH in which some degradation is observed, are successful inliberating free peptide from APS-modified peptide. Additional studiesare carried out with incubation at 55° C. or 85° C. for one hour.APS-modified peptide is equally stable at 55° C. and is only slightlyless stable at 85° C. Some acid hydrolysis, indicated by the presence ofnovel peaks in the HPLC chromatogram, is observed with the 1M HCl sampleincubated at 85° C. for one hour.

Example 15 Purification of APS-Modified MBI 11CN

[0264] A large scale preparation of APS-modified MBI 11CN is purified.Approximately 400 mg of MBI 11CN is APS-modified and dissolved in 20 mlof water. Unreacted MBI 11CN is removed by RP-HPLC. The solvent is thenevaporated from the APS-modified MBI 11CN pool, and the residue isdissolved in 10 ml methylene chloride. The modified peptide is thenprecipitated with 10 ml diethyl ether. After 5 min at ambienttemperature, the precipitate is collected by centrifugation at 5000×gfor 10 minutes. The pellet is washed with 5 ml of diethyl ether andagain collected by centrifugation at 5000×g for 10 minutes. Thesupernatants are pooled for analysis of unreacted polysorbateby-products. The precipitate is dissolved in 6 ml of water and thenflushed with nitrogen by bubbling for 30 minutes to remove residualether. The total yield from the starting MBI 11CN was 43%.

Example 16 Biological Assays Using APS-Modified Peptide

[0265] All biological assays that compare APS-modified peptides withunmodified peptides are performed on an equimolar ratio. Theconcentration of APS-modified peptides can be determined byspectrophotometric measurement, which is used to normalizeconcentrations for biological assays. For example, a 1 mg/mlAPS-modified MBI 11CN solution contains the same amount of peptide as a1 mg/ml MBI 11CN solution, thus allowing direct comparison of toxicityand efficacy data.

[0266] APS-modified peptides are at least as potent as the parentpeptides in in vitro assays performed as described herein. MIC valuesagainst gram positive bacteria are presented for several APS-modifiedpeptides and compared with the values obtained using the parent peptides(Table 22). The results indicate that the modified peptides are at leastas potent in vitro as the parent peptides and may be more potent thanthe parent peptides against E. faecalis strains. TABLE 22 Corrected MIC(μg/ml) Organism Organism # Peptide APS-peptide Peptide A. calcoaceticusAC002 MBI 11B1CN 4 2 A. calcoaceticus AC002 MBI 11B7CN 8 4 A.calcoaceticus AC002 MBI 11CN >64 4 A. calcoaceticus AC002 MBI 11E3CN 8 2A. calcoaceticus AC002 MBI 11F3CN 8 2 E. cloacae ECL007 MBI 11B1CN128 >128 E. cloacae ECL007 MBI 11B7CN 128 128 E. cloacae ECL007 MBI 11CN64 >128 E. cloacae ECL007 MBI 11E3CN 128 >128 E. cloacae ECL007 MBI11F3CN 128 >128 E. coli ECO005 MBI 11B1CN 16 8 E. coli ECO005 MBI 11B7CN64 8 E. coli ECO005 MBI 11CN 64 16 E. coli ECO005 MBI 11E3CN 64 8 E.coli ECO005 MBI 11F3CN 128 16 E. faecalis EFS001 MBI 11B1CN 4 32 E.faecalis EFS001 MBI 11B7CN 8 8 E. faecalis EFS001 MBI 11CN 8 32 E.faecalis EFS001 MBI 11E3CN 4 8 E. faecalis EFS001 MBI 11F3CN 8 32 E.faecalis EFS004 MBI 11B1CN 4 8 E. faecalis EFS004 MBI 11B7CN 8 8 E.faecalis EFS004 MBI 11CN 4 8 E. faecalis EFS004 MBI 11E3CN 4 2 E.faecalis EFS004 MBI 11F3CN 4 16 E. faecalis EFS008 MBI 11B1CN 8 32 E.faecalis EFS008 MBI 11B7CN 8 32 E. faecalis EFS008 MBI 11CN 64 64 E.faecalis EFS008 MBI 11E3CN 8 16 E. faecalis EFS008 MBI 11F3CN 4 128 K.pneumoniae KP001 MBI 11B1CN 32 128 K. pneumoniae KP001 MBI 11B7CN 64 16K. pneumoniae KP001 MBI 11CN 64 128 K. pneumoniae KP001 MBI 11E3CN 64 8K. pneumoniae KP001 MBI 11F3CN 128 64 P. aeruginosa PA004 MBI 11B1CN 128128 P. aeruginosa PA004 MBI 11B7CN 128 128 P. aeruginosa PA004 MBI 11CN64 >128 P. aeruginosa PA004 MBI 11E3CN 128 128 P. aeruginosa PA004 MBI11F3CN 128 128 S. aureus SA010 MBI 11B1CN 4 1 S. aureus SA010 MBI 11B7CN4 1 S. aureus SA010 MBI 11CN 4 2 S. aureus SA010 MBI 11E3CN 2 1 S.aureus SA010 MBI 11F3CN 4 2 S. aureus SA011 MBI 11B1CN 16 4 S. aureusSA011 MBI 11B7CN 16 4 S. aureus SA011 MBI 11CN 16 8 S. aureus SA011 MBI11E3CN 16 4 S. aureus SA011 MBI 11F3CN 16 8 S. aureus SA014 MBI 11B1CN 48 S. aureus SA014 MBI 11B7CN 8 4 S. aureus SA014 MBI 11CN 8 16 S. aureusSA014 MBI 11E3CN 4 4 S. aureus SA014 MBI 11F3CN 8 8 S. aureus SA018 MBI11B1CN 32 16 S. aureus SA018 MBI 11B7CN 32 16 S. aureus SA018 MBI 11CN64 64 S. aureus SA018 MBI 11E3CN 32 16 S. aureus SA018 MBI 11F3CN 64 16S. aureus SA025 MBI 11B1CN 4 1 S. aureus SA025 MBI 11B7CN 2 1 S. aureusSA025 MBI 11CN 2 4 S. aureus SA025 MBI 11E3CN 2 1 S. aureus SA025 MBI11F3CN 4 2 S. aureus SA093 MBI 11B1CN 2 1 S. aureus SA093 MBI 11B7CN 2 1S. aureus SA093 MBI 11CN 2 2 S. aureus SA093 MBI 11E3CN 2 1 S. aureusSA093 MBI 11F3CN 2 1 S. maltophilia SMA002 MBI 11B1CN 64 128 S.maltophilia SMA002 MBI 11B7CN 128 32 S. maltophilia SMA002 MBI 11CN >64128 S. maltophilia SMA002 MBI 11E3CN 128 64 S. maltophilia SMA002 MBI11F3CN 128 64 S. marcescens SMS003 MBI 11B1CN 128 >128 S. marcescensSMS003 MBI 11B7CN 128 >128 S. marcescens SMS003 MBI 11CN 64 >128 S.marcescens SMS003 MBI 11E3CN 128 >128 S. marcescens SMS003 MBI 11F3CN128 >128

[0267] Toxicities of APS-modified MBI 11CN and unmodified MBI 11CN areexamined in Swiss CD-1 mice. Groups of 6 mice are injected iv withsingle doses of 0.1 ml peptide in 0.9% saline. The dose levels used are0, 3, 5, 8, 10, and 13 mg/kg. Mice are monitored at 1, 3, and 6 hrspost-injection for the first day, then twice daily for 4 days. Thesurvival data for MBI 11CN mice are presented in Table 23. ForAPS-modified MBI 11CN, 100% of the mice survived at all doses, includingthe maximal dose of 13 mg/kg. TABLE 23 Peptide administered CumulativeNo. Cumulative No. (mg/kg) No. Dead/Total Dead Surviving Dead/Total %Dead 13 6/6 18 0 18/18 100 10 6/6 12 0 12/12 100 8 6/6 6 0 6/6 100 5 0/60 6 0/6 0 3 0/6 0 12 0/12 0 0 0/6 0 18 0/18 0

[0268] As summarized below, the LD₅₀ for MBI 11CN is 7 mg/kg (Table 24),with all subjects dying at a dose of 8 mg/ml. The highest dose of MBI11CN giving 100% survival was 5 mg/kg. The data show that APS-modifiedpeptides are significantly less toxic than the parent peptides. TABLE 24Test Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI-11CN-TFA  7 mg/kg  8 mg/kg  5 mg/kgAPS-MBI-11CN >13 mg/kg* >13 mg/kg* >13 mg/kg*

[0269] It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

1 90 8 amino acids amino acid single linear Modified-site 2 /note=“Wherein X is a Hydrophobic Residue” Modified-site 3 /note= “Wherein Xis Proline or Valine” Modified-site 4 /note= “Wherein X is a HydrophobicResidue” Modified-site 5 /note= “Wherein X is a Hydrophobic Residue”Modified-site 6 /note= “Wherein X is Proline or Valine” Modified-site 7/note= “Wherein X is a Hydrophobic Residue” Modified-site 8 /note=“Wherein X is a Basic Amino Acid” 1 Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 58 amino acids amino acid single linear Modified-site 1 /note= “Wherein Xis a Basic Amino Acid” Modified-site 2 /note= “Wherein X is aHydrophobic Residue” Modified-site 3 /note= “Wherein X is Proline orValine” Modified-site 4 /note= “Wherein X is a Hydrophobic Residue”Modified-site 5 /note= “Wherein X is a Hydrophobic Residue”Modified-site 6 /note= “Wherein X is Proline or Valine” Modified-site 7/note= “Wherein X is a Hydrophobic Residue” Modified-site 8 /note=“Wherein X is a Basic Amino Acid” 2 and 6 /note= “At least One of theResidues at Positions 2 or 6 is Valine” 2 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 amino acids amino acid single linear Modified-site 1 /note=“Wherein X is a Basic Amino Acid” Modified-site 2 /note= “Wherein X is aBasic Amino Acid” Modified-site 3 /note= “Wherein X is a Basic AminoAcid” Modified-site 4 /note= “Wherein X is a Hydrophobic Residue”Modified-site 5 /note= “Wherein X is Proline or Valine” Modified-site 6/note= “Wherein X is a Hydrophobic Residue” Modified-site 7 /note=“Wherein X is a Hydrophobic Residue” Modified-site 8 /note= “Wherein Xis Proline or Valine” Modified-site 9 /note= “Wherein X is a HydrophobicResidue” Modified-site 10 /note= “Wherein X is a Basic Amino Acid” 3 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 17 amino acids amino acidsingle linear Modified-site 1 /note= “Wherein X is a Basic Amino Acid”Modified-site 2 /note= “Wherein X is a Hydrophobic Amino Acid”Modified-site 3 /note= “Wherein X is Proline or Valine” Modified-site 4/note= “Wherein X is a Hydrophobic Residue” Modified-site 5 /note=“Wherein X is a Hydrophobic Residue” Modified-site 6 /note= “Wherein Xis Proline or Valine” Modified-site 7 /note= “Wherein X is a HydrophobicResidue” Modified-site 8 /note= “Wherein X is a Basic Amino Acid”Modified-site 9 /note= “Wherein X is a Basic Amino Acid” Modified-site13 /note= “Wherein X is a Basic Amino Acid” Modified-site 14 /note=“Wherein X is a Basic Amino Acid” 4 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaMet Ile Leu Xaa Xaa Ala Gl 1 5 10 15 Ser 18 amino acids amino acidsingle linear Modified-site 1 /note= “Wherein X is a Basic Amino Acid”Modified-site 2 /note= “Wherein X is a Hydrophobic Amino Acid”Modified-site 3 /note= “Wherein X is Proline or Valine” Modified-site 4/note= “Wherein X is a Hydrophobic Residue” Modified-site 5 /note=“Wherein X is a Hydrophobic Residue” Modified-site 6 /note= “Wherein Xis Proline or Valine” Modified-site 7 /note= “Wherein X is a HydrophobicResidue” Modified-site 8 /note= “Wherein X is a Basic Amino Acid”Modified-site 9 /note= “Wherein X is a Basic Amino Acid” Modified-site10 /note= “Wherein X is a Basic Amino Acid” Modified-site 14 /note=“Wherein X is a Basic Amino Acid” Modified-site 15 /note= “Wherein X isa Basic Amino Acid” 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met IleLeu Xaa Xaa Al 1 5 10 15 Gly Ser 18 amino acids amino acid single linearModified-site 1 /note= “Wherein X is a Basic Amino Acid” Modified-site 2/note= “Wherein X is a Hydrophobic Amino Acid” Modified-site 3 /note=“Wherein X is Proline or Valine” Modified-site 4 /note= “Wherein X is aHydrophobic Residue” Modified-site 5 /note= “Wherein X is a HydrophobicResidue” Modified-site 6 /note= “Wherein X is Proline or Valine”Modified-site 7 /note= “Wherein X is a Hydrophobic Residue”Modified-site 8 /note= “Wherein X is a Basic Amino Acid” Modified-site 9/note= “Wherein X is a Basic Amino Acid” Modified-site 14 /note=“Wherein X is a Basic Amino Acid” Modified-site 15 /note= “Wherein X isa Basic Amino Acid” 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met IleLeu Xaa Xaa Al 1 5 10 15 Gly Ser 19 amino acids amino acid single linearModified-site 1 /note= “Wherein X is a Basic Amino Acid” Modified-site 2/note= “Wherein X is a Hydrophobic Amino Acid” Modified-site 3 /note=“Wherein X is Proline or Valine” Modified-site 4 /note= “Wherein X is aHydrophobic Residue” Modified-site 5 /note= “Wherein X is a HydrophobicResidue” Modified-site 6 /note= “Wherein X is Proline or Valine”Modified-site 7 /note= “Wherein X is a Hydrophobic Residue”Modified-site 8 /note= “Wherein X is a Basic Amino Acid” Modified-site 9/note= “Wherein X is a Basic Amino Acid” Modified-site 10 /note=“Wherein X is a Basic Amino Acid” Modified-site 15 /note= “Wherein X isa Basic Amino Acid” Modified-site 16 /note= “Wherein Xaa is a BasicAmino Acid” 7 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Ile LeuXaa Xa 1 5 10 15 Ala Gly Ser 10 amino acids amino acid single linearModified-site 1 /note= “Wherein X is a Basic Amino Acid” Modified-site 2/note= “Wherein X is a Hydrophobic Amino Acid” Modified-site 3 /note=“Wherein X is Proline or Valine” Modified-site 4 /note= “Wherein X is aHydrophobic Residue” Modified-site 5 /note= “Wherein X is a HydrophobicResidue” Modified-site 6 /note= “Wherein X is Proline or Valine”Modified-site 7 /note= “Wherein X is a Hydrophobic Residue”Modified-site 8 /note= “Wherein X is a Basic Amino Acid” Modified-site 9/note= “Wherein X is a Basic Amino Acid” 8 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Met 1 5 10 11 amino acids amino acid single linear Modified-site1 /note= “Wherein X is a Basic Amino Acid” Modified-site 2 /note=“Wherein X is a Hydrophobic Amino Acid” Modified-site 3 /note= “WhereinX is Proline or Valine” Modified-site 4 /note= “Wherein X is aHydrophobic Residue” Modified-site 5 /note= “Wherein X is a HydrophobicResidue” Modified-site 6 /note= “Wherein X is Proline or Valine”Modified-site 7 /note= “Wherein X is a Hydrophobic Residue”Modified-site 8 /note= “Wherein X is a Basic Amino Acid” Modified-site 9/note= “Wherein X is a Basic Amino Acid” 9 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Met 1 5 10 8 amino acids amino acid single linearModified-site 2 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site3 /note= “Wherein Xaa is a Hydrophobic Residue” Modified-site 4 /note=“Wherein Xaa is a Hydrophobic Residue” Modified-site 5 /note= “WhereinXaa is a Hydrophobic Residue” Modified-site 6 /note= “Wherein Xaa is aHydrophobic Residue” 10 Leu Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 9 aminoacids amino acid single linear Modified-site 2 /note= “Wherein Xaa is aBasic Amino Acid” Modified-site 3 /note= “Wherein Xaa is a Basic AminoAcid” Modified-site 4 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 5 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 6 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 7 /note= “Wherein Xaa is a Hydrophobic Residue” 11 Leu XaaXaa Xaa Xaa Xaa Xaa Arg Lys 1 5 9 amino acids amino acid single linearModified-site 2 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site3 /note= “Wherein Xaa is a Hydrophobic Residue” Modified-site 4 /note=“Wherein Xaa is Proline or Valine” Modified-site 5 /note= “Wherein Xaais a Hydrophobic Residue” Modified-site 6 /note= “Wherein Xaa is aHydrophobic Residue” Modified-site 7 /note= “Wherein Xaa is aHydrophobic Residue” 12 Leu Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 9 aminoacids amino acid single linear Modified-site 2 /note= “Wherein Xaa is aBasic Amino Acid” Modified-site 3 /note= “Wherein Xaa is a HydrophobicResidue” Modified-site 4 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 5 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 6 /note= “Wherein Xaa is Proline or Valine” Modified-site7 /note= “Wherein Xaa is a Hydrophobic Residue” 13 Leu Xaa Xaa Xaa XaaXaa Xaa Arg Lys 1 5 10 amino acids amino acid single linearModified-site 2 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site3 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site 4 /note=“Wherein Xaa is a Hydrophobic Residue” Modified-site 5 /note= “WhereinXaa is Proline or Valine” Modified-site 6 /note= “Wherein Xaa is aHydrophobic Residue” Modified-site 7 /note= “Wherein Xaa is aHydrophobic Residue” Modified-site 8 /note= “Wherein Xaa is aHydrophobic Residue” 14 Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 1010 amino acids amino acid single linear Modified-site 2 /note= “WhereinXaa is a Basic Amino Acid” Modified-site 3 /note= “Wherein Xaa is aBasic Amino Acid” Modified-site 4 /note= “Wherein Xaa is a HydrophobicResidue” Modified-site 5 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 6 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 7 /note= “Wherein Xaa is Proline or Valine” Modified-site8 /note= “Wherein Xaa is a Hydrophobic Residue” 15 Leu Xaa Xaa Xaa XaaXaa Xaa Xaa Arg Lys 1 5 10 10 amino acids amino acid single linearModified-site 2 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site3 /note= “Wherein Xaa is a Hydrophobic Residue” Modified-site 4 /note=“Wherein Xaa is Proline or Valine” Modified-site 5 /note= “Wherein Xaais a Hydrophobic Residue” Modified-site 6 /note= “Wherein Xaa is aHydrophobic Residue” Modified-site 7 /note= “Wherein Xaa is Proline orValine” Modified-site 8 /note= “Wherein Xaa is a Hydrophobic Residue” 16Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 10 11 amino acids amino acidsingle linear Modified-site 2 /note= “Wherein Xaa is a Basic Amino Acid”Modified-site 3 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site4 /note= “Wherein Xaa is a Hydrophobic Residue” Modified-site 5 /note=“Wherein Xaa is Proline or Valine” Modified-site 6 /note= “Wherein Xaais a Hydrophobic Residue” Modified-site 7 /note= “Wherein Xaa is aHydrophobic Residue” Modified-site 8 /note= “Wherein Xaa is Proline orValine” Modified-site 9 /note= “Wherein Xaa is a Hydrophobic Residue” 17Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 10 10 amino acids aminoacid single linear Modified-site 2 /note= “Wherein Xaa is a HydrophobicResidue” Modified-site 3 /note= “Wherein Xaa is Proline or Valine”Modified-site 4 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 5 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 6 /note= “Wherein Xaa is Proline or Valine” Modified-site7 /note= “Wherein Xaa is a Hydrophobic Residue” 18 Leu Xaa Xaa Xaa XaaXaa Xaa Arg Arg Lys 1 5 10 11 amino acids amino acid single linearModified-site 3 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 4 /note= “Wherein Xaa is Proline or Valine” Modified-site5 /note= “Wherein Xaa is a Hydrophobic Residue” Modified-site 6 /note=“Wherein Xaa is a Hydrophobic Residue” Modified-site 7 /note= “WhereinXaa is Proline or Valine” Modified-site 8 /note= “Wherein Xaa is aHydrophobic Residue” 19 Leu Lys Xaa Xaa Xaa Xaa Xaa Xaa Arg Arg Lys 1 510 11 amino acids amino acid single linear Modified-site 1 /note=“Wherein Xaa is a Basic Amino Acid” Modified-site 2 /note= “Wherein Xaais a Basic Amino Acid” Modified-site 3 /note= “Wherein Xaa is aHydrophobic Residue” Modified-site 4 /note= “Wherein Xaa is Proline orValine” Modified-site 5 /note= “Wherein Xaa is a hydrophobic Residue”Modified-site 6 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 7 /note= “Wherein Xaa is Proline or Valine” Modified-site8 /note= “Wherein Xaa is a Hydrophobic Residue” Modified-site 9 /note=“Wherein Xaa is a Basic Amino Acid” Modified-site 10 /note= “Wherein Xaais a Basic Amino Acid” Modified-site 11 /note= “Wherein Xaa is a BasicAmino Acid” 3,5,6 and 8 /note= “At least Two of the Residues atPositions 3,5,6 and 8 are Phenyalanine” 20 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 1 5 10 11 amino acids amino acid single linearModified-site 1 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site2 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site 3 /note=“Wherein Xaa is a Hydrophobic Residue” Modified-site 4 /note= “WhereinXaa is Proline or Valine” Modified-site 5 /note= “Wherein Xaa is ahydrophobic Residue” Modified-site 6 /note= “Wherein Xaa is aHydrophobic Residue” Modified-site 7 /note= “Wherein Xaa is Proline orValine” Modified-site 8 /note= “Wherein Xaa is a Hydrophobic Residue”Modified-site 9 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site10 /note= “Wherein Xaa is a Basic Amino Acid” Modified-site 11 /note=“Wherein Xaa is a Basic Amino Acid” Modified-site 3,5,6 and 8 /note= “Atleast Two of the Residues at Positions 3,5,6 and 8 are Tyrosine” 21 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 13 amino acids amino acid<Unknown> linear 22 Ile Leu Lys Lys Phe Pro Phe Phe Pro Phe Arg Arg Lys1 5 10 13 amino acids amino acid <Unknown> linear 23 Ile Leu Lys Lys TyrPro Tyr Tyr Pro Tyr Arg Arg Lys 1 5 10 12 amino acids amino acid<Unknown> linear 24 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Lys 1 510 13 amino acids amino acid <Unknown> linear 25 Ile Leu Arg Arg Trp ProTrp Trp Pro Trp Arg Arg Arg 1 5 10 13 amino acids amino acid <Unknown>linear 26 Trp Arg Ile Trp Lys Pro Lys Trp Arg Leu Pro Lys Trp 1 5 10 12amino acids amino acid <Unknown> linear 27 Ile Leu Arg Trp Val Trp TrpVal Trp Arg Arg Lys 1 5 10 11 amino acids amino acid <Unknown> linear 28Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Lys 1 5 10 13 amino acids aminoacid <Unknown> linear 29 Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp ArgArg 1 5 10 13 amino acids amino acid <Unknown> linear 30 Ile Leu Lys LysTrp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 13 amino acids amino acid<Unknown> linear 31 Lys Arg Arg Trp Pro Trp Trp Pro Trp Lys Lys Leu Ile1 5 10 13 amino acids amino acid <Unknown> linear 32 Ile Leu Lys Lys IlePro Ile Ile Pro Ile Arg Arg Lys 1 5 10 12 amino acids amino acid<Unknown> linear 33 Ile Leu Lys Lys Trp Pro Trp Pro Trp Arg Arg Lys 1 510 13 amino acids amino acid <Unknown> linear 34 Ile Leu Lys Lys Tyr ProTrp Tyr Pro Trp Arg Arg Lys 1 5 10 13 amino acids amino acid <Unknown>linear 35 Ile Leu Lys Lys Phe Pro Trp Phe Pro Trp Arg Arg Lys 1 5 10 13amino acids amino acid <Unknown> linear 36 Ile Leu Lys Lys Phe Pro PheTrp Pro Trp Arg Arg Lys 1 5 10 12 amino acids amino acid <Unknown>linear 37 Ile Leu Arg Tyr Val Tyr Tyr Val Tyr Arg Arg Lys 1 5 10 13amino acids amino acid <Unknown> linear 38 Ile Leu Arg Arg Trp Pro TrpTrp Pro Trp Arg Arg Lys 1 5 10 12 amino acids amino acid <Unknown>linear 39 Ile Leu Arg Arg Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10 12amino acids amino acid <Unknown> linear 40 Ile Leu Lys Trp Pro Trp TrpPro Trp Arg Arg Lys 1 5 10 11 amino acids amino acid <Unknown> linear 41Ile Leu Lys Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10 12 amino acids aminoacid <Unknown> linear 42 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys1 5 10 12 amino acids amino acid <Unknown> linear 43 Lys Arg Arg Trp ProTrp Trp Pro Trp Arg Leu Ile 1 5 10 11 amino acids amino acid <Unknown>linear 44 Ile Leu Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 13 aminoacids amino acid <Unknown> linear 45 Ile Leu Lys Lys Trp Pro Trp Trp ProTrp Lys Lys Lys 1 5 10 21 amino acids amino acid <Unknown> linear 46 IleLeu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Ile Met Ile Le 1 5 10 15 LysLys Ala Gly Ser 20 20 amino acids amino acid <Unknown> linear 47 Ile LeuArg Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Leu Ly 1 5 10 15 Lys AlaGly Ser 20 21 amino acids amino acid <Unknown> linear 48 Ile Leu Arg TrpPro Trp Trp Pro Trp Arg Arg Lys Asp Met Ile Le 1 5 10 15 Lys Lys Ala GlySer 20 13 amino acids amino acid <Unknown> linear 49 Ile Leu Lys Lys TrpAla Trp Trp Pro Trp Arg Arg Lys 1 5 10 13 amino acids amino acid<Unknown> linear 50 Ile Leu Lys Lys Trp Pro Trp Trp Ala Trp Arg Arg Lys1 5 10 13 amino acids amino acid <Unknown> linear 51 Trp Trp Lys Lys TrpPro Trp Trp Pro Trp Arg Arg Lys 1 5 10 12 amino acids amino acid<Unknown> linear 52 Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 510 8 amino acids amino acid <Unknown> linear 53 Pro Trp Trp Pro Trp ArgArg Lys 1 5 21 amino acids amino acid <Unknown> linear 54 Ile Leu LysLys Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Le 1 5 10 15 Lys Lys AlaGly Ser 20 20 amino acids amino acid <Unknown> linear 55 Ile Leu Lys LysTrp Pro Trp Trp Pro Trp Arg Arg Met Ile Leu Ly 1 5 10 15 Lys Ala Gly Ser20 21 amino acids amino acid <Unknown> linear 56 Ile Leu Lys Lys Trp ProTrp Trp Pro Trp Arg Arg Ile Met Ile Le 1 5 10 15 Lys Lys Ala Gly Ser 2014 amino acids amino acid <Unknown> linear 57 Ile Leu Lys Lys Trp ProTrp Trp Pro Trp Arg Arg Lys Met 1 5 10 13 amino acids amino acid<Unknown> linear 58 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Met1 5 10 14 amino acids amino acid <Unknown> linear 59 Ile Leu Lys Lys TrpPro Trp Trp Pro Trp Arg Arg Ile Met 1 5 10 11 amino acids amino acid<Unknown> linear 60 Ile Leu Lys Lys Trp Trp Trp Pro Trp Arg Lys 1 5 1011 amino acids amino acid <Unknown> linear 61 Ile Leu Lys Lys Trp ProTrp Trp Trp Arg Lys 1 5 10 13 amino acids amino acid <Unknown> linearModified-site 1 /note= “D-Form of Isoleucine” 62 Ile Leu Lys Lys Trp ProTrp Trp Pro Trp Arg Arg Lys 1 5 10 13 amino acids amino acid <Unknown>linear Modified-site 13 /note= “D-Form of Lysine” 63 Ile Leu Lys Lys TrpPro Trp Trp Pro Trp Arg Arg Lys 1 5 10 13 amino acids amino acid<Unknown> linear Modified-site 1 /note= “D-Form of Isoleucine”Modified-site 13 /note= “D-Form of Lysine” 64 Ile Leu Lys Lys Trp ProTrp Trp Pro Trp Arg Arg Lys 1 5 10 13 amino acids amino acid <Unknown>linear 65 Ile Leu Lys Lys Trp Val Trp Trp Val Trp Arg Arg Lys 1 5 10 13amino acids amino acid <Unknown> linear 66 Ile Leu Lys Lys Trp Pro TrpTrp Val Trp Arg Arg Lys 1 5 10 13 amino acids amino acid <Unknown>linear 67 Ile Leu Lys Lys Trp Val Trp Trp Pro Trp Arg Arg Lys 1 5 10 12amino acids amino acid <Unknown> linear 68 Lys Arg Arg Trp Val Trp TrpVal Trp Arg Leu Ile 1 5 10 12 amino acids amino acid <Unknown> linear 69Ile Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 12 amino acidsamino acid <Unknown> linear 70 Ile Leu Lys Lys Pro Trp Trp Pro Trp ArgArg Lys 1 5 10 12 amino acids amino acid <Unknown> linear 71 Ile Leu LysLys Trp Trp Trp Pro Trp Arg Arg Lys 1 5 10 12 amino acids amino acid<Unknown> linear 72 Ile Leu Lys Lys Trp Pro Trp Trp Trp Arg Arg Lys 1 510 12 amino acids amino acid <Unknown> linear 73 Ile Leu Lys Lys Trp ProTrp Trp Pro Arg Arg Lys 1 5 10 12 amino acids amino acid <Unknown>linear 74 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10 11amino acids amino acid <Unknown> linear 75 Ile Leu Lys Lys Trp Pro TrpTrp Pro Trp Arg 1 5 10 12 amino acids amino acid <Unknown> linear 76 AlaLeu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 12 amino acids aminoacid <Unknown> linear 77 Ile Ala Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys1 5 10 12 amino acids amino acid <Unknown> linear 78 Ile Leu Ala Trp ProTrp Trp Pro Trp Arg Arg Lys 1 5 10 12 amino acids amino acid <Unknown>linear 79 Ile Leu Arg Ala Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 12amino acids amino acid <Unknown> linear 80 Ile Leu Arg Trp Ala Trp TrpPro Trp Arg Arg Lys 1 5 10 12 amino acids amino acid <Unknown> linear 81Ile Leu Arg Trp Pro Ala Trp Pro Trp Arg Arg Lys 1 5 10 12 amino acidsamino acid <Unknown> linear 82 Ile Leu Arg Trp Pro Trp Ala Pro Trp ArgArg Lys 1 5 10 12 amino acids amino acid <Unknown> linear 83 Ile Leu ArgTrp Pro Trp Trp Ala Trp Arg Arg Lys 1 5 10 12 amino acids amino acid<Unknown> linear 84 Ile Leu Arg Trp Pro Trp Trp Pro Ala Arg Arg Lys 1 510 12 amino acids amino acid <Unknown> linear 85 Ile Leu Arg Trp Pro TrpTrp Pro Trp Ala Arg Lys 1 5 10 12 amino acids amino acid <Unknown>linear 86 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Ala Lys 1 5 10 12amino acids amino acid <Unknown> linear 87 Ile Leu Arg Trp Pro Trp TrpPro Trp Arg Arg Ala 1 5 10 7 amino acids amino acid <Unknown> linear 88Trp Trp Pro Trp Arg Arg Lys 1 5 7 amino acids amino acid <Unknown>linear 89 Ile Leu Lys Lys Trp Pro Trp 1 5 9 amino acids amino acid<Unknown> linear 90 Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5

We claim:
 1. An indolicidin analogue, comprising 8 to 25 amino acids and containing the formula: RXZXXZXBwherein Z is proline or valine; X is a hydrophobic residue; and B is a basic amino acid.
 2. An indolicidin analogue, comprising 8 to 25 amino acids and containing the formula: BXZXXZXBwherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; and wherein at least one Z is valine.
 3. An indolicidin analogue, comprising 10 to 25 amino acids and containing the formula: BBBXZXXZXBwherein Z is proline or valine; X is a hydrophobic residue; and B is a basic amino acid.
 4. An indolicidin analogue, comprising 17 to 25 amino acids and containing the formula: BXZXXZXBBB_(n)(AA)_(n)MILBBAGSwherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; AA is any amino acid, and n is 0 or
 1. 5. An indolicidin analogue, comprising 10 to 25 amino acids and containing the formula: BXZXXZXBB(AA)_(n)Mwherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; AA is any amino acid, and n is 0 or
 1. 6. An indolicidin analogue, comprising 8 to 25 amino acids and containing the formula: LBB_(n)XZ_(n)XXZ_(n)XRKwherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; and n is 0 or
 1. 7. An indolicidin analogue, comprising 10 to 25 amino acids and containing the formula: LK_(n)XZXXZXRRKwherein Z is proline or valine; X is a hydrophobic residue; and n is 0 or
 1. 8. An indolicidin analogue, comprising 11 to 25 amino acids and containing the formula: BBXZXXZXBBBwherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; and wherein at least two X residues are phenylalanine.
 9. An indolicidin analogue, comprising 11 to 25 amino acids and containing the formula: BBXZXXZXBBBwherein Z is proline or valine; X is a hydrophobic residue; B is a basic amino acid; and wherein at least two X residues are tyrosine.
 10. The analogues according to any one of claims 1 and 3-7 wherein Z is proline, X is tryptophan and B is arginine or lysine.
 11. An indolicidin analogue selected from the group consisting of: Lys Arg Arg Trp Pro Trp Trp Pro Trp Lys Lys Leu Ile; Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys; Lys Arg Arg Trp Pro Trp Trp Pro Trp Arg Leu Ile; Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Ile Met Ile Leu Lys Lys Ala Gly Ser; Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Leu Lys Lys Ala Gly Ser; Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Asp Met Ile Leu Lys Lys Ala Gly Ser; Trp Arg Ile Trp Lys Pro Lys Trp Arg Leu Pro Lys Trp; Ile Leu Lys Lys Trp Val Trp Trp Pro Trp Arg Arg Lys; Ile Leu Arg Trp Val Trp Trp Val Trp Arg Arg Lys; and Lys Arg Arg Trp Val Trp Trp Val Trp Arg Leu Ile;


12. An indolicidin analogue selected from the group consisting of: Ile Leu Lys Lys Ile Pro Ile Ile Pro Ile Arg Arg Lys; Ile Leu Lys Lys Tyr Pro Tyr Tyr Pro Tyr Arg Arg Lys; Ile Leu Lys Lys Tyr Pro Trp Tyr Pro Trp Arg Arg Lys; Ile Leu Lys Lys Phe Pro Trp Phe Pro Trp Arg Arg Lys; Ile Leu Lys Lys Phe Pro Phe Trp Pro Trp Arg Arg Lys; Ile Leu Arg Tyr Val Tyr Tyr Val Tyr Arg Arg Lys; Ile Leu Arg Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Arg Arg Trp Pro Trp Trp Pro Trp Arg Lys; Ile Leu Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Lys; Ile Leu Lys Trp Pro Trp Trp Pro Trp Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Met Ile Lou Lys Lys Ala Gly Ser; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Ile Met Ile Leu Lys Lys Ala Gly Ser; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Met; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Ile Met; Ile Leu Lys Lys Trp Trp Trp Pro Trp Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Trp Arg Lys; Ile Leu Lys Lys Trp Vol Trp Trp Val Trp Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Val Trp Arg Arg Lys;


13. An indolicidin analogue selected from the group consisting of: Ile Leu Trp Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp Pro Trp Arg Arg Lys; Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys; Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Leu Lys Lys Ala Gly Ser; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys Met; Trp Trp Pro Trp Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp; Ile Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Lys Lys Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Lys Lys Trp Trp Trp Pro Trp Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Trp Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Pro Arg Arg Lys; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Lys; Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg; Trp Pro Trp Trp Pro Trp Arg Arg Lys;


14. An indolicidin analogue selected from the group consisting of: Ala Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys; Ile Ala Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Ala Trp Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Arg Ala Pro Trp Trp Pro Trp Arg Arg Lys; Ile Leu Arg Trp Ala Trp Trp Pro Trp Arg Arg Lys; Ile Leu Arg Trp Pro Ala Trp Pro Trp Arg Arg Lys; Ile Leu Arg Trp Pro Trp Ala Pro Trp Arg Arg Lys; Ile Leu Arg Trp Pro Trp Trp Ala Trp Arg Arg Lys; Ile Leu Arg Trp Pro Trp Trp Pro Ala Arg Arg Lys; Ile Leu Arg Trp Pro Trp Trp Pro Trp Ala Arg Lys; Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Ala Lys; Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Ala;


15. The indolicidin analogue according to any one of claims 1-14, wherein two or more analogues are coupled to form a branched peptide.
 16. The indolicidin analogue according to any one of claims 1 to 14, wherein the analogue has one or more amino acids altered to a corresponding D-amino acid.
 17. The indolicidin analogue according to claim 16, wherein the N-terminal and/or the C-terminal amino acid is a D-amino acid.
 18. The indolicidin analogue according to any one of claims 1-14, wherein the analogue is acetylated at the N-terminal amino acid.
 19. The indolicidin analogue according to any one of claims 1-14, wherein the analogue is amidated at the C-terminal amino acid.
 20. The indolicidin analogue according to any one of claims 1-14, wherein the analogue is esterified at the C-terminal amino acid.
 21. The indolicidin analogue according to any one of claims 1-14, wherein the analogue is modified by incorporation of homoserine/homoserine lactone at the C-terminal amino acid.
 22. The indolicidin analogue according to any one of claims 1-14, wherein the analogue is conjugated with polyethylene glycol or derivatives thereof.
 23. An isolated nucleic acid molecule whose sequence comprises one or more coding sequences of an indolicidin analogue according to any one of claims 11-14.
 24. An expression vector comprising a promoter in operable linkage with the nucleic acid molecule of claim
 23. 25. A host cell transfected or transformed with the expression vector of claim
 24. 26. A pharmaceutical composition comprising at least one indolicidin analogue according to any of claims 1-22 and a physiologically acceptable buffer.
 27. The pharmaceutical composition according to claim 26, further comprising an antiviral agent, an antiparasitic agent, or an antifungal agent.
 28. The pharmaceutical composition according to claim 26, further comprising an antibiotic agent.
 29. The pharmaceutical composition according to claim 28, wherein the antibiotic is selected from the group consisting of penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, quinolones, tetracyclines, aminoglycosides, macrolides, glycopeptides, chloramphenicols, glycylcyclines, licosamides and fluoroquinolones.
 30. The pharmaceutical composition according to claim 28, wherein the antibiotic is selected from the group consisting of Amikacin; Amoxicillin; Ampicillin; Azithromycin; Azlocillin; Aztreonam; Carbenicillin; Cefaclor; Cefamandole formate sodium; Cefazolin; Cefepime; Cefetamet; Cefixime; Cefmetazole; Cefonicid; Cefoperazone; Cefotaxime; Cefotetan; Cefoxitin; Cefpodoxime; Cefprozil; Cefsulodin; Ceftazidime; Ceftizoxime; Ceftriaxone; Cefuroxime; Cephalexin; Cephalothin; Chloramphenicol; Cinoxacin; Ciprofloxacin; Clarithromycin; Clindamycin; Cloxacillin; Co-amoxiclavulanate; Dicloxacillin; Doxycycline; Enoxacin; Erythromycin; Erythromycin estolate; Erythromycin ethyl succinate; Erythromycin glucoheptonate; Erythromycin lactobionate; Erythromycin stearate; Ethambutol; Fleroxacin; Gentamicin; Imipenem; Isoniazid; Kanamycin; Lomefloxacin; Loracarbef,; Meropenem Methicillin; Metronidazole; Mezlocillin; Minocycline hydrochloride; Mupirocin; Nafcillin; Nalidixic acid; Netilmicin; Nitrofurantoin; Norfloxacin; Ofloxacin; Oxacillin; Penicillin G; Piperacillin; Pyrazinamide; Rifabutin; Rifampicin; Roxithromycin; Streptomycin; Sulfamethoxazole; Synercid; Teicoplanin; Tetracycline; Ticarcillin; Tobramycin; Trimethoprim; Vancomycin; a combination of Piperacillin and Tazobactam; and derivatives thereof.
 31. The pharmaceutical composition according to claim 28, wherein the antibiotic is selected from the group consisting of Amikacin; Azithromycin; Cefoxitin; Ceftriaxone; Ciprofloxacin; Co-amoxiclavulanate; Doxycycline; Gentamicin; Mupirocin; Vancomycin; and a combination of Piperacillin and Tazobactam.
 32. A pharmaceutical composition comprising a physiologically acceptable buffer and a combination of an analogue and an antibiotic, wherein the combination is selected from the group consisting of: I L K K F P F F P F R R K and Ciprofloxacin; I L K K F P F F P F R R K and Mupirocin; I L K K Y P Y Y P Y R R K and Mupirocin; I L K K W P W W P W R K and Mupirocin; I L R R W P W W P W R R R and Piperacillin; W R I W K P K W R L P K W and Ciprofloxacin; W R I W K P K W R L P K W and Mupirocin; W R I W K P K W R L P K W and Piperacillin; I L R W V W W V W R R K and Piperacillin; and I L K K W P W W P W K and Mupirocin.
 33. A method of treating an infection, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to any of claims 26-32.
 34. The method of claim 33, wherein the infection is due to a microorganism.
 35. The method of claim 34, wherein the microorganism is selected from the group consisting of bacterium, fungus, parasite and virus.
 36. The method of claim 35, wherein the fungus is a yeast and/or mold.
 37. The method of claim 35, wherein the parasite is selected from the group consisting of protozoan, nematode, cestode and trematode.
 38. The method of claim 35, wherein the bacterium is a Gram-negative bacterium.
 39. The method of claim 38, wherein the Gram-negative bacterium is selected from the group consisting of Acinetobacter spp., Enterobacter spp., E. coli, H. influenzae, K. pneumoniae, P. aeruginosa, S. marcescens, S. maltophilia Bordetella pertussis, Brucella spp., Campylobacter spp., Haemophilus ducreyi, Helicobacter pylori, Legionella spp., Moraxella catarrhalis, Neisseria spp., Salmonella spp., Shigella spp. and Yersinia spp.
 40. The method of claim 35, wherein the bacterium is a Gram-positive bacterium.
 41. The method of claim 40, wherein the Gram-positive bacterium is selected from the group consisting of E. faecalis, S. aureus, E. faecium, S. pyogenes, S. pneumoniae, coagulase-negative staphylococci, Bacillus spp., Corynebacterium spp., Propionibacterium acne, Diphtheroids, Listeria spp. and Viridans Streptococci.
 42. The method of claim 35, wherein the bacterium is an anaerobe.
 43. The method of claim 42, wherein the anaerobe is selected from the group consisting of Clostridium spp., Bacteroides spp. and Peptostreptococcus spp., Borrelia spp., Chlamydia spp., Mycobacterium spp., Mycoplasma spp., Rickettsia spp., Treponema spp. and Ureaplasma spp.
 44. The method of claim 33, wherein the analogue is administered by intravenous injection, intraperitoneal injection or implantation, intramuscular injection or implantation, intrathecal injection, subcutaneous injection or implantation, intradernal injection, lavage, bladder wash-out, suppositories, pessaries, oral ingestion, topical application, enteric application, inhalation, aerosolization or nasal spray or drops.
 45. A composition, comprising an indolicidin analogue according to any of claims 1-14 and an antibiotic.
 46. A device coated with a composition comprising the indolicidin analogue according to claims 1-26.
 47. The device of claim 46, wherein the composition further comprises an antibiotic agent.
 48. The device of claim 46, wherein the device is a medical device.
 49. An antibody that reacts specifically with the analogue according to any of claims 11-14.
 50. A composition comprising a compound modified by derivatization of an amino group with a conjugate comprising activated polyoxyalkylene glycol and a fatty acid.
 51. The composition of claim 50, wherein the conjugate further comprises sorbitan linking the polyoxyalkylene glycol and fatty acid.
 52. The composition of claim 50, wherein the conjugate is polysorbate.
 53. The composition of claim 50, wherein the polyoxyalkylene glycol is polyoxyethylene.
 54. The composition of claim 50, wherein the compound is a peptide or protein.
 55. The composition of claim 54, wherein the peptide is indolicidin or an indolicidin analogue.
 56. The composition of claim 50, wherein the polyoxyalkylene glycol is activated by irradiation with ultraviolet light.
 57. A method of making a compound modified with a conjugate of an activated polyoxyalkylene glycol and a fatty acid, comprising: (a) freezing a mixture of the conjugate of an activated polyoxyalkylene glycol and fatty acid with the compound; and (b) lyophilizing the frozen mixture; wherein the compound has a free amino group.
 58. The method of claim 57, wherein the compound is a peptide or an antibiotic.
 59. The method of claim 57, wherein the mixture in step (a) is in an acetate buffer.
 60. A method of making a compound modified with a conjugate of an activated polyoxyalkylene glycol and a fatty acid, comprising mixing the conjugate of an activated polyoxyalkylene glycol and fatty acid with the compound; for a time sufficient to form modified compounds, wherein the mixture is in a carbonate buffer having a pH greater than 8.5 and the compound has a free amino group.
 61. The method of claim 60, wherein the compound is a peptide or an antibiotic.
 62. The method of either of claims 57 or 60, further comprising isolating the modified compound by reversed-phase HPLC and/or by precipitation of the modified compound from an organic solvent.
 63. A pharmaceutical composition comprising at least one composition according to any of claims 50-62 and a physiologically acceptable buffer
 64. The pharmaceutical composition according to claim 63, further comprising an antibiotic agent, an antiviral agent, an antiparasitic agent or an antifungal agent.
 65. A method of treating an infection, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to any of claims 63-64.
 66. The method of claim 65, wherein the infection is due to a microorganism selected from the group consisting of bacterium, fungus, parasite and virus. 